Nucleic acid and amino acid sequences relating to mycobacterium tuberculosis and leprae for diagnostics and therapeutics

ABSTRACT

Embodiments of the present invention feature nucleic acid and proteins derived from  Mycobacterium tuberculosis  and  leprae . The proteins and nucleic acid of the present invention have applications in diagnostics and therapeutics.

This application is a continuation-in-part of U.S. Ser. No. 08/109,181filed Aug. 19, 1993, now abandoned, and U.S. Ser. No. 08/142,558 filedOct. 22, 1993, now abandoned, the disclosure of which is incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to non-naturally occurring nucleic acidand peptides corresponding to nucleic acid and peptides of Mycobacteriumtuberculosis and Mycobacterium leprae. The nucleic acid and peptides ofthe present invention have utility for diagnostics and therapeutics.

BACKGROUND OF THE INVENTION

Mycobacterium tuberculosis is the causative agent of tuberculosis.Tuberculosis is a chronic bacterial infection characterized by theformation of granulomas in infected tissues and by cell mediatedhypersensitivity. The usual site of the disease is the lungs but otherorgans may be involved. In countries where human immuno-deficiency virus(HIV) infection is endemic, tuberculosis is a frequent cause ofmorbidity in AIDS patients. Tuberculosis has shown a resurgence inrecent years worldwide. Mycobacteria contain an array of protein andpolysaccharide antigens giving rise to a cell mediated hypersensitivity.The hypersensitivity is often used to diagnose tuberculosis and tomonitor the disease pathogenesis. See: Harrison's Principles of InternalMedicine, Twelfth Edition, McGraw-Hill, Inc., (1991), pp. 637-645.

Mycobacterium leprae is the causative agent of Hanson's disease orleprosy. Leprosy is a chronic infection of superficial tissues,especially the skin and peripheral nerves. Mycobacterium lepraemultiplies slowly, and has not been grown in tissue culture orartificial media. Mycobacterium leprae does not elicit strongimmunological responses in infected individuals. However, aserodiagnostic test for the detection of antibody to Mycobacteriumleprae antigen is used to aid in the diagnosis. See Harrison, supra, pp.645-648.

Patient care, as well as the prevention and transmission ofMycobacterium tuberculosis and leprae, requires reliable diagnostic andprognostic tools to detect nucleic acid, antigens and antibodiesrelating to tuberculosis and leprosy.

A number of therapeutic agents are currently available for combattingMycobacterium tuberculosis and Mycobacterium leprae infections in man.However, limitations to these therapies, particularly for Mycobacteriumtuberculosis infection, demonstrate that new, more effective agents areneeded. For example, agents such as isoniazid and rifampicin arecurrently in use for Mycobacterium tuberculosis infections, but strainswhich are resistant to one or the other or both of these agents arebeing reported with increasing frequency (eg., Chawla et al., 1992, Am.Rev. Respir. Dis. 146, 278-279). In addition, Mycobacterium tuberculosisbacilli treated by drug therapy frequently remain viable, but dormant aslatent infections in macrophage phagolysosomes which may re-emerge asfull-blown tuberculosis at a later date (eg., Dannenberg, in “TheMycobacteria: A Sourcebook”, Kubica and Wayne, eds., Dekker, N.Y., pp.721-760).

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to diagnostics and therapeutics for Mycobacteriumtuberculosis, M. leprae and other mycobacterial species including, butnot limited to: M. avium, M. bovis, M. chitae, M. fortuitum, M.gorodonae, M. intracellulare, M. kansaii, M. paratuberculosis, M.smegmatis, M. terrae, and M. ulcerans. One embodiment of the presentinvention features, as an article of manufacture or as a composition, anon-naturally occurring nucleic acid having twenty or more nucleotidesin a sequence corresponding to a sequence of Mycobacterium tuberculosisor Mycobacterium leprae nucleic acid. The non-naturally occurringnucleic acid of the present invention is exemplified by Seq. ID No. 1with respect to Mycobacterium tuberculosis and by Seq. ID Nos. 23-26 and120-140 with respect to Mycobacterium leprae. Preferably, the sequencehas at least twenty, and more preferably at least thirty nucleotides ina sequence corresponding to Mycobacterium tuberculosis or Mycobacteriumleprae nucleic acid. The sequence may correspond to the entire codingsequence for the gene or a part of the coding sequence. However,sequences larger than 1000 nucleotides in length are difficult tosynthesize.

One embodiment of the present invention features a non-naturallyoccurring nucleic acid wherein the nucleic acid encodes a peptide ofMycobacterium tuberculosis or M. leprae or other mycobacterial species.The peptide has utility for diagnostics and therapeutics.

A further embodiment of the present invention comprises a non-naturallyoccurring nucleic acid wherein the nucleic acid is capable of bindingmessenger RNA of Mycobacterium tuberculosis or M. leprae or othermycobacterial species. Such non-naturally occurring nucleic acid iscapable of acting as anti-sense nucleic acid to control the translationof messenger RNA of Mycobacterium tuberculosis, M. leprae or othermycobacterial species. The non-naturally occurring nucleic acid inhibitsthe translation of Mycobacterium tuberculosis, M. leprae or othermycobacterial gene products, which by way of example, relate to thesynthesis of antibiotics and the constituents of the cell wall,intermediary metabolism, transport processes, nucleic acid biosynthesisand modification, and antibiotic resistance. One of ordinary skill inthe art would readily be able to discern which proteins were involved inthe above metabolic processes. The above listed processes, as well asthe proteins involved in such processes, are well described in anycellular biology textbook such as Molecular Cell Biology, Darnell, J.,Lodish, H., and Baltimore, D. Scientific American Books, N.Y. Suchnon-naturally occurring nucleic acids have utility in therapeutics anddiagnostics. A further embodiment of the present invention features anon-naturally occurring nucleic acid which nucleic acid is capable ofbinding specifically to Mycobacterium tuberculosis or leprae nucleicacid. Such non-naturally occurring nucleic acid has utility as probesand as capture reagents.

One embodiment of the present invention features an expression systemcomprising an open reading frame of DNA corresponding to Mycobacteriumtuberculosis or leprae DNA. The nucleic acid further comprises a controlsequence compatible with an intended host. The expression system isuseful for making peptides corresponding to Mycobacterium tuberculosisor leprae nucleic acid.

A further embodiment of the present invention features a celltransformed with the expression system to make Mycobacteriumtuberculosis or leprae proteins and peptides.

Several non-naturally occurring nucleic acids of the present inventionfeature nucleic acid relating to products involved in cell wallbiosynthesis of Mycobacterium tuberculosis. Such nucleic acids havesequences of twenty or more nucleotides which correspond to sequences ofSeq. ID No. 1. One such nucleic acid has a sequence of twenty or morenucleotides which correspond to a sequence within nucleotides 28,325 to31,285 of Seq. ID No. 1. Nucleotides 28,325 to 31,285 encode a gene forpolyketide or fatty acid synthesis. The gene codes for an enzyme withacyl transferase, enoyl reductase and dehydratase domains. The putativeamino acid sequence of the gene product is set forth in Seq. ID No. 2.

An enzyme, ketoacyl ACP synthase of Mycobacterium tuberculosis, isencoded by nucleotides 26750 to 28237 of Seq. ID No. 1. The putativeamino acid sequence of the enzyme is set forth in Seq. ID No. 3.

Nucleic acid encoding a gene for the enzyme beta-keto reductase ofMycobacterium tuberculosis corresponds to nucleotides 24636 to 26753 ofSeq. ID No. 1. The putative amino acid sequence is set forth in Seq. IDNo. 4.

Nucleic acid encoding COA ligases correspond to nucleotides 22136 to23371 or 23251 to 23994 of Seq. ID No. 1. The putative amino acidsequence is set forth in Seq. ID No. 5 and Seq. ID No. 118.

Nucleic acids encoding two UDP-sugar transferases of Mycobacteriumtuberculosis corresponds to nucleotides 9489 to 10846 of Seq. ID No. 1and 12604 to 13995 of Seq. ID No. 1. The putative amino acid sequenceare set forth in Seq. ID Nos. 6 and 7.

Nucleic acid encoding a methyltransferase of Mycobacterium tuberculosiscorresponds to nucleotides 20006 to 19347 of Seq. ID No. 1. The putativeamino acid sequence is set forth in Seq. ID No. 8.

Nucleic acid encoding a KdtB protein of Mycobacterium tuberculosiscorresponds to nucleotides 5790 to 6275 of Seq. ID No. 1. KdtB proteinis an essential 12 kD protein associated with the synthesis oflipopolysaccharide. The putative amino acid sequence is set forth inSeq. ID No. 9.

A further non-naturally occurring nucleic acid of the present inventionhas a sequence of twenty nucleotides which correspond to proteinsinvolved in intermediary metabolism. One non-naturally occurring nucleicacid corresponds to a pyruvate carboxylase of Mycobacteriumtuberculosis. Pyruvate carboxylase is involved in gluconeogenesis. Theenzyme catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetatein the presence of cofactors biotin and zinc. The nucleotides 1565 to4939 of Seq. ID No. 1 encode the enzyme pyruvate carboxylase. Theputative amino acid sequence of the enzyme is set forth in Seq. ID No.10.

One non-naturally occurring nucleic acid has a sequence of twenty ormore nucleotides corresponding to a phosphoribosylglycinamideformyltransferase. Phosphoribosylglycinamide formyltransferase catalyzesthe third step in de novo purine biosynthesis. The nucleotides encodingphosphoribosylglycinamide formyltransferase are 8061 to 7144 of Seq. IDNo. 1. The putative amino acid sequence of the enzyme is set forth inSeq. ID No. 11.

A further non-naturally occurring nucleic acid of the present inventioncorresponds to gene products involved in transport processes. Onenon-naturally occurring nucleic acid encodes a structural gene for ananion pump protein of Mycobacterium tuberculosis. The nucleic acid has asequence which corresponds to a sequence within nucleotides 9369 to 8149of Seq. ID No. 1 which encode the pump. The putative amino acid sequenceis set forth in Seq. ID No. 12.

A further non-naturally occurring nucleic acid has a sequence of twentyor more nucleotides which correspond to gene products involved in DNAbiosynthesis and cell division. One non-naturally occurring nucleic acidof the present invention corresponds to a Mycobacterium tuberculosiscell division protein. A Mycobacterium tuberculosis cell divisionprotein is encoded at nucleotide positions 5042 to 5704 of Seq. IDNo. 1. The putative amino acid sequence is set forth in Seq. ID No. 13.

One embodiment of the present invention features a non-naturallyoccurring nucleic acid having a sequence of twenty or more nucleotideswhich correspond to gene products that are involved in antibioticresistance. One non-naturally occurring nucleic acid of the presentinvention corresponds to a ribosomal RNA methylase. The nucleic acid hasa sequence of twenty or more nucleotides which corresponds to a sequencewithin nucleotides 17223 to 17933 of Seq. ID No. 1 encoding for theenzyme. The putative amino acid sequence is set forth in Seq. ID No. 14.

A further non-naturally occurring nucleic acid has a sequence of twentyor more nucleotides which correspond to a sequence of Mycobacteriumtuberculosis useful as a probe to identify Mycobacterium tuberculosisgenes or homologous genes in other mycobacteria or other relatedbacterial species. Nucleic acid sequences that are specific toMycobacterium tuberculosis genes correspond to nucleotides 15841 to15203, nucleotides 15131 to 14306, nucleotides 20491 to 21489,nucleotides 911 to 1540, nucleotides 16223 to 17161, nucleotides 24020to 24619, nucleotides 3 to 902, nucleotides 11313 to 11651, andnucleotides 11766 to 12503. The putative amino acid sequences are setforth in Seq. ID Nos. 15, 16, 17, 18, 19, 20, 21, 22, 118 and 119. Theseamino acid sequences may define proteins which are specific toMycobacterium tuberculosis. Such proteins may give rise to antibodieswhich are specific to Mycobacterium tuberculosis.

The Mycobacterium tuberculosis peptides, the functions of such peptides,and the nucleotide positions associated with such peptides within Seq.ID No. 1 are summarized in Table I below.

TABLE I Enzyme name or activity Gene Position (SeqID No.) Function ermK17223-17933 ribosomal RNA methylase :resistance (# 14) gltA  9489-10846putative UDP-sugar :synth:carbo transferase (# 6) gltB 12604-13995putative UDP-sugar :synth:carbo transferase (# 7) kdtB 5790-6275 12 kDa;lipopolysaccharide :synth:lipid biosynth (# 9) ligA 22136-23371 CoAligase subunit (# 5) :synth:lipid ligB 23251-23994 CoA ligase subunit (#118) :synth:lipid mtrA 20006-19347 putative methyl transferase :synth (#8) pksA 28325-31285 polyketide synthase; AT, ER, :synth:lipid DH (# 2)pksB 26750-28237 polyketide synthase; AS (# 3) :synth:lipid pksC24636-26753 polyketide synthase; KR (# 4) :synth:lipid pur3 8061-7144phosphoribosylglycinamide :synth:nt formyltr..(# 11) pycA 1565-4939pyruvate carboxylase (# 10) :metab ttbc2a 9369-8149 anion pump protein(# 12) :transport yhhF 5042-5704 cell division protein (# 13) :celldivision utbc2a 15841-15203 M. tuberculosis gene :M.tb-specific sequence(# 15) utbc2b 15131-14306 M. tuberculosis gene :M.tb-specific sequence(# 16) utbc2c 20491-21489 M. tuberculosis gene :M.tb-specific sequence(# 17) utbc2d  911-1540 M. tuberculosis gene :M.tb-specific sequence (#18) utbc2e 16223-17161 M. tuberculosis gene :M.tb-specific sequence (#19) utbc2f 24020-24619 M. tuberculosis gene :M.tb-specific sequence (#20) utbc2g  3-902 M. tuberculosis gene :M.tb-specific sequence (# 21)utbc2h 11313-11651 M. tuberculosis gene :M.tb-specific sequence (# 22)utbc2i 11766-12503 M. tuberculosis gene :M.tb-specific sequence (# 119)

Tables I-XXVI contain information on the genes encoded by the cosmidsequences with the corresponding Seq. ID Nos. (see table headings).Tables I-XIX consist of four columns with the heading gene, position,enzyme name or activity with Seq. ID No., and function. Tables XX-XXVIconsist of five columns with the heading gene, SegID, position, enzymeor protein name, and function. Under the heading “gene”, a specific nameis provided where the gene is positively identified by homology to otherorganisms, or a letter followed by the cosmid number. The letter “d”represents dehydrogenase, “t” represents transport associated, “a”represents ATPase. Under the heading “SeqID”, a specific sequence IDnumber is provided. Under the heading “position”, a range of nucleotideswith the first and last numbers corresponding to the start and stoppositions are identified which correspond to the respective Seq. ID No.In tables I-V, the start positions correspond to probable translationinitiation codons; in tables VI-XXVI, the start positions correspond tothe beginning of the reading frame which includes the predictable invivo start site but usually is some distance away. Under the heading“Enzyme name or activity”, an exact name may be given, an activity(e.g., dehydrogenase), an indication of homology to other sequences inpublic databases, or the designation—M. leprae gene sequence, indicatinga previously un-described gene. Under the heading “function”, a numberof functional categories, as described in the text are identified; forexample, “metab” denotes intermediary metabolism. In some casesmetabolism is more specifically broken down in the following manner;“synth” denotes biosynthesis, “catab” denotes breakdown. Furtherinformation is sometimes given on the compounds involved; “lipid”denotes lipid, “aa” denotes amino acid, “nt” denotes nucleotide, “carbo”denotes carbohydrate, “glyco” denotes glycolysis, “antibiotic” denotesantibiotics, “protein” denotes “protein”, “tca” denotes tricarboxylicacid cycle, “wall” denotes cell wall, “cofact” denotes cofactor (anindication of the specific cofactor sometimes follows), “M. tb-specific”denotes an M. tuberculosis gene useful as a probe, “M. leprae-specific”denotes an M. leprae gene useful as a probe, “transport” denotes a geneinvolved in transport, cell division denotes a gene involved in “celldivision”, “resistance” denotes a gene implicated in antibioticresistance, “repair” denotes a DNA repair gene, “cell div.” denotes agene involved in cell division, “antigen” denotes a gene that is knownto be a mycobacterial antigen, or an antigen in another organism thatmay be useful for vaccine development, “redox” denotes involved inelectron transport, “stress” denotes a gene that is involved in cellularstress responses, “regulatory” denotes regulatory gene, “translation”denotes a gene whose product is involved in ribosome function, and“recombination” denotes a gene involved in recombination.

Several non-naturally occurring nucleic acids of the present inventionfeature nucleic acid relating to products of Mycobacterium Leprae. Onenon-naturally occurring nucleic acid of the present invention has asequence of twenty or more nucleotides which correspond to a sequencewithin Seq.

ID Nos. 23-26 and 120-140. The Mycobacterium Leprae peptides, thefunctions of such peptides, and nucleotide positions associated withsuch peptides within Seq. ID No. 23 are summarized in Table II below.

Table II Enzyme name or activity Gene Position (SeqID No.) Function ybaB32443-32093 12 kD protein (# 27) :M. leprae-specific dhaS 23517-22465aspartate-semialdehyde :metab. dehydrog. (# 28) glpK 14121-12658glycerol kinase (# 29) :metab. akaB 24783-23520 aspartokinase (# 30):metab. recM 32078-31470 recM/recR (# 31) :repair:cell div. leu125583-27298 2-isopropylmalate :metab. synthase (# 32) pbpA 3032-5494penicillin binding :resistance protein (# 33) arsA 1060-1914anion-transporting :transport ATPase (# 34) u0577a  38-1063 M. lepraegene :M.leprae-specific sequence (# 35) u0577b 1911-2204 M. leprae gene:M.leprae-specific sequence (# 37) u0577c 5513-6622 M. leprae gene:M.leprae-specific sequence (# 38) u0577d 7161-7474 M. leprae gene:M.leprae-specific sequence (# 39) u0577e 10765-11355 M. leprae gene:M.leprae-specific sequence (# 40) u0577f 11789-12388 M. leprae gene:M.leprae-specific sequence (# 41) u0577g 14034-14276 M. leprae gene:M.leprae-specific sequence (# 42) u0577h 14269-14880 M. leprae gene:M.leprae-specific sequence (# 43) u0577i 15548-15922 M. leprae gene:M.leprae-specific sequence (# 44) u0577j 28664-29812 M. leprae gene:M.leprae-specific sequence (# 45) u0577k 29805-30512 M. leprae gene:M.leprae-specific sequence (# 46) u0577l 33851-35230 M. leprae gene:M.leprae-specific sequence (# 47) u0577m 35227-36546 M. leprae gene:M.leprae-specific sequence (# 48) u0577n 18608-17988 M. leprae gene:M.leprae-specific sequence (# 49) u0577o 22453-21305 M. leprae gene:M.leprae-specific sequence (# 50) u0577p 33762-33325 M. leprae gene:M.leprae-specific sequence (# 51)

Nucleotides 32443 to 32093 of Seq. ID No. 23 encode a hypothetical 12 kDprotein in the DNAX-RECR intergenic region. The putative amino acidsequence is set forth in Seq. ID No. 27.

One non-naturally occurring nucleic acid of the present inventioncorresponds to a sequence encoding the enzyme aspartate-semialdehydedehydrogenase. The enzyme, aspartate-semialdehyde dehydrogenase, isinvolved in intermediary metabolism and is encoded by nucleotides 23517to 22465 of Seq. ID No. 23. The putative amino acid sequence is setforth in Seq. ID No. 28.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding the enzyme glycerolkinase. The enzyme, glycerol kinase, is involved in intermediarymetabolism. The nucleotides encoding glycerol kinase are 14121 to 12658of Seq. ID No. 23. The putative amino acid sequence is set forth in Seq.ID No. 29.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding the enzyme aspartatekinase. The enzyme, aspartate kinase, is involved in intermediarymetabolism. The nucleotide sequence encoding aspartate kinase are 24783to 23520 of Seq. ID No. 23. The putative amino acid sequence is setforth in Seq. ID No. 30.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding the RecM protein. Thisprotein is involved in DNA repair DNA replication and cell division. Thenucleotides encoding RecM protein are 32078 to 31470 of Seq. ID No. 23.The putative amino acid sequence is set forth in Seq. ID No. 31.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds a sequence encoding to 2-isopropyl malatesynthase. The enzyme, 2-isopropyl malate synthase, is involved inintermediary metabolism. The nucleotides encoding 2-isopropyl malatesynthase are 25583 to 27298 of Seq. ID No. 23. The putative amino acidsequence is set forth in Seq. ID No. 32.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a penicillin bindingprotein, involved in antibiotic resistance. The nucleotides encoding thepenicillin binding protein are 3032 to 5494 of Seq. ID No. 23. Theputative amino acid sequence is set forth in Seq. ID No. 33.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding an anion-transportingATPase involved in transport processes. The nucleotides encoding thistransport protein are 1060 to 1914 of Seq. ID No. 23. The putative aminoacid sequence is set forth in Seq. ID No. 34.

Non-naturally occurring nucleic acids having a sequence of twenty ormore nucleotides which correspond to a sequence of Mycobacterium lepraeare useful as probes to identify Mycobacterium leprae genes orhomologous genes in other mycobacteria or other related bacterialspecies. Nucleic acid sequences that are specific to Mycobacteriumleprae genes correspond to nucleotides 38 to 1063, nucleotides 1060 to1917, nucleotides 1911 to 2204, nucleotides 5513 to 6622, nucleotides7161 to 7474, nucleotides 10765 to 11355, nucleotides 11789 to 12388,nucleotides 14034 to 14276, nucleotides 14269 to 14880, nucleotides15548 to 15922, nucleotides 28664 to 29812, nucleotides 29805 to 30512,nucleotides 33851 to 35230, nucleotides 35227 to 36546, nucleotides18608 to 17988, nucleotides 22453 to 21305, nucleotides 33762 to 33325of Seq. ID No. 23. The respective putative amino acid sequences are setforth in Seq. ID Nos. 35-51.

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptide within Seq. ID No. 24are summarized in Table III set forth below.

TABLE III Enzyme name or activity Gene Position (SeqID No.) Functionf1912a 16941-18311 CoA ligase modifying :metab. enzyme (# 52) dhpS28531-29322 dihydropteroate synthase :metab. (fol. acid) (# 53) 3mg130740-31315 dna-3-methyladenine :metab, :repair glycosidase (# 54) glgC33059-34315 glucose-1-P- :synth:carbo adenylyltransferase (# 55) u1912a 693-2900 68 kD tpp-requiring :metab. protein (# 56) ag45 20486-21097 45kD serine-rich :antigen antigen (# 57) dcuP 7954-9044 uroporphyrinogen:metab. decarboxylase (# 58) u1912b 4637-5104 M. leprae gene:M.leprae-specific sequence (# 59) u1912c 25695-26399 M. leprae gene:M.leprae-specific sequence (# 60) u1912d 26195-26758 M. leprae gene:M.leprae-specific sequence (# 61) u1912e 27975-28217 M. leprae gene:M.leprae-specific sequence (# 62) u1912f 29399-30484 M. leprae gene:M.leprae-specific sequence (# 63) u1912g 30396-30740 M. leprae gene:M.leprae-specific sequence (# 64) u1912h 30737-31315 M. leprae gene:M.leprae-specific sequence (# 65) u1912i 31589-31756 M. leprae gene:M.leprae-specific sequence (# 66) u1912j 34389-35042 M. leprae gene:M.leprae-specific sequence (# 67) u1912k 6290-5001 M. leprae gene:M.leprae-specific sequence (# 68) u1912l 6882-6292 M. leprae gene:M.leprae-specific sequence (# 69) u1912m  9041-10396 M. leprae gene:M.leprae-specific sequence (# 70) u1912n 19930-18315 M. leprae gene:M.leprae-specific sequence (# 71) u1912o 23399-22446 M. leprae gene:M.leprae-specific sequence (# 72) u1912p 32508-32260 M. leprae gene:M.leprae-specific sequence (# 73) u1912q 10402-11097 M. leprae gene:M.leprae-specific sequence (# 74) u1912r 35946-35617 M. leprae gene:M.leprae-specific sequence (# 75) u1912s 12935-13234 M. leprae gene:M.leprae-specific sequence (# 76) u1912t 13486-13779 M. leprae gene:M.leprae-specific sequence (# 77) u1912u 21186-21488 M. leprae gene:M.leprae-specific sequence (# 78) u1912v 21540-21827 M. leprae gene:M.leprae-specific sequence (# 79) u1912w 22002-22355 M. leprae gene:M.leprae-specific sequence (# 80) u1912x 23315-24517 M. leprae gene:M.leprae-specific sequence (# 81)

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a CoA ligase modifyingenzyme. The enzyme, CoA ligase, is involved in intermediary metabolism.The nucleotides encoding this CoA ligase are 16941 to 18311 of Seq. IDNo. 24. The putative amino acid sequence is set forth in Seq. ID No. 52.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding dihydropteroatesynthase (DHPS). The enzyme, dihydropteroate synthase is involved inintermediary metabolism. The nucleotides encoding DHPS are 28531 to29322 of Seq. ID No. 24. The putative amino acid sequence is set forthin Seq. ID No. 53.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding DNA-3-methyladenineglycosidase I. The enzyme, DNA-3-methyladenine glycosidase I, isinvolved in intermediary metabolism. The nucleotides encoding thisglycosidase are 30740 to 31315 of Seq. ID No. 24. The putative aminoacid sequence is set forth in Seq. ID No. 54.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding glucose-1-phosphateadenyltransferase. The enzyme, glucose-1-phosphate adenyltransferase, isinvolved in intermediary metabolism. The nucleotides encoding thisenzyme are 33059 to 34315 of Seq. ID No. 24. The putative amino acidsequence is set forth in Seq. ID No. 55.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a 68 kD thiaminepyrophosphate-requiring protein. The 68 kD enzyme is involved inintermediary metabolism. The nucleotides encoding this protein are 693to 2900 of Seq. ID No. 24. The putative amino acid sequence is set forthin Seq. ID No. 56.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a probable antigenidentified in Mycobacterium tuberculosis. The nucleotides encoding thisprobable antigen are 20486 to 21097 of Seq. ID No. 24. The putativeamino acid sequence is set forth in Seq. ID No. 57.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding uroporphyrinogendecarboxylase. The enzyme, uroporphyrinogen decarboxylase, is involvedin intermediary metabolism. The nucleotides encoding this enzyme are7954 to 9044 of Seq. ID No. 24. The putative amino acid sequence is setforth in Seq. ID No. 58.

Non-naturally occurring nucleic acids having a sequence of twenty ormore nucleotides which correspond to a sequence of Mycobacterium lepraeare useful as a probe to identify Mycobacterium leprae genes orhomologous genes in other mycobacteria or other related bacterialspecies. Nucleic acid sequences that are specific to Mycobacteriumleprae genes include nucleotides at positions 4637 to 5104, nucleotides25695 to 26399, nucleotides 26195 to 26758, nucleotides 27995 to 28217,nucleotides 29399 to 30484, nucleotides 30396 to 30740, nucleotides30737 to 31315, nucleotides 31589 to 31756, nucleotides 34389 to 35042,nucleotides 6290 to 5001, nucleotides 6882 to 6292, nucleotides 9041 to10396, nucleotides 19930 to 18315, nucleotides 23399 to 22446,nucleotides 32508 to 32260, nucleotides 10402 to 11097, nucleotides35946 to 35617, nucleotides 12935 to 13234, nucleotides 13486 to 13779,nucleotides 21186 to 21488, nucleotides 21540 to 21827, nucleotides22002 to 22355, and nucleotides 23315 to 24517 of Seq. ID No. 24. Theputative amino acid sequences are set forth in Seq. ID Nos. 59-81.

Mycobacterium leprae peptides, the function of such peptides, thenucleotide positions associated with, such peptides within Seq. ID No.25 are summarized in Table IV set forth below.

TABLE IV Enzyme name or activity Gene Position (SeqID No.) Function adhA3301-4332 alcohol dehydrogenase (# 82) :metab. fbp 26808-25829fibronectin binding :antigen:stress prot./85-B Ag (# 83) ahpC12402-11818 alkyl hydroperoxide :antigen:redox reductase C (# 84) bfr15629-16105 bacterioferritin (# 85) :redox adhT 28957-28664 alcoholdehydrogenase (# 86) :metab. cysT 139-396 sulfate permease (# 87):transport cpx 22311-22078 cytochrome P450 :synth: hydroxylase (# 88)antibiotic: oxyR 12485-13447 inducible regulatory :metab. protein (# 89)gox 9872-8631 2-hydroxy-acid :metab oxidase (# 90) u0038a 24755-24309 M.leprae gene sequence :M.leprae- (# 92) specific u0038b 34108-33197 M.leprae gene sequence :M.leprae- (# 93) specific u0038c 717-859 M. lepraegene sequence :M.leprae- (# 94) specific u0038d 1047-1268 M. leprae genesequence :M.leprae- (# 95) specific u0038e 1928-2791 M. leprae genesequence :M.leprae- (# 96) specific u0038f 2951-3250 M. leprae genesequence :M.leprae- (# 97) specific u0038g 10854-11063 M. leprae genesequence :M.leprae- (# 98) specific u0038h 5683-4985 M. leprae genesequence :M.leprae- (# 99) specific u0038i 10433-10176 M. leprae genesequence :M.leprae- (# 100) specific u0038j 24266-23821 M. leprae genesequence :M.leprae- (# 101) specific

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding alcohol dehydrogenase(ADH). The enzyme, alcohol dehydrogenase, is involved in intermediarymetabolism and detoxification. The nucleotides encoding ADH are 3301 to4332 of Seq. ID No. 25. The putative amino acid sequence is set forth inSeq. ID No. 82.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding the Mycobacteriumleprae 85-B antigen (alpha antigen). The nucleotides associated with85-B antigen are 26808 to 25829 of Seq. ID No. 25. The putative aminoacid sequence is set forth in Seq. ID No. 83.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a major mycobacterialantigen and the enzyme alkyl hydroperoxide reductase (AHPC). The enzyme,mycobacterial antigen, is a detoxifying enzyme. The nucleotides encodingAHPC are 12402 to 11818 of Seq. ID No. 25. The putative amino acidsequence is set forth in Seq. ID No. 84.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding bacterioferritin(BFR). Bacterioferritin is involved in iron detoxification and storage.The nucleotides encoding BFR are 15629 to 16105 of Seq. ID No. 25. Theputative amino acid sequence is set forth in Seq. ID No. 85.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding alcoholdehydrogenase-T (ADH-T). The enzyme, alcohol dehydrogenase-T, isinvolved in intermediary metabolism. The nucleotides encoding ADH-T are28957 to 28664 of Seq. ID No. 25. The putative amino acid sequence isset forth in Seq. ID No. 86.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a molybdenum orsulfate transport protein. The nucleotides encoding this transportprotein are located at positions 139 to 396 of Seq. ID No. 25. Theputative amino acid sequence is set forth in Seq. ID No. 87.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a cytochrome P450-likehydroxylase. This enzyme is involved in detoxification processes orantibiotic synthesis. The nucleotides encoding this protein are 22311 to22078 of Seq. ID No. 25. The putative amino acid sequence is set forthin Seq. ID No. 88.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a hydrogen peroxideinducible regulatory protein (OXYR). The nucleotides OXYR are 12485 to13447 of Seq. ID No. 25. The putative amino acid sequence is set forthin Seq. ID No. 89.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding an enzyme(S)-2-hydroxy-acid oxidase (GOX). The enzyme, GOX is involved inintermediary metabolism. The nucleotides encoding GOX are 9872 to 8631of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq.ID No. 290.

Non-naturally occurring nucleic acids having a sequence of twenty ormore nucleotides which correspond to a sequence of Mycobacterium lepraeare useful as probes to identify Mycobacterium leprae genes orhomologous genes in other mycobacteria or other related bacterialspecies. Nucleic acid sequences that are specific to Mycobacteriumleprae genes include nucleotides at positions 139 to 396, nucleotides24755 to 24309, nucleotides 34108 to 33197, nucleotides 717 to 859,nucleotides 1047 to 1268, nucleotides 1928 to 2791, nucleotides 2951 to3250, nucleotides 10854 to 11063, nucleotides 5683 to 4985, nucleotides10433 to 10176, and nucleotides 24266 to 23821 of Seq. ID No. 25. Theputative amino acid sequences are set forth in Seq. ID Nos. 91-101.

Mycobacterium leprae peptides, the function of such peptides and thesequences associated with such peptides within Seq. ID No. 26 aresummarized in Table V set forth below.

TABLE V Enzyme name or activity Gene Position (SeqID No.) Function cspA27760-28176 cold-shock protein (# 102) :stress erc 20362-19211 DNAhelicase excision :DNA repair repair (# 103) fadA 7672-8880 fatty acidoxidase beta :metab. subunit (# 104) fadB  8855-11029 fatty acid oxidasealpha :metab. subunit (# 105) dciP 4228-2522 indolepyruvate :metab.decarboxylase (# 106) u1935a 37098-36712 putative Ca-binding :metab.protein (# 107) u1935b 4745-5074 M. leprae gene sequence:M.leprae-specific (# 108) u1935c 35805-34525 M. leprae gene sequence:M.leprae-specific (# 109) u1935d 16602-17078 M. leprae gene sequence:M.leprae-specific (# 110) u1935e 36372-37181 M. leprae gene sequence:M.leprae-specific (# 111) u1935f 6871-6071 M. leprae gene sequence:M.leprae-specific (# 112) u1935g 20856-20293 M. leprae gene sequence:M.leprae-specific (# 113) u1935h 21696-20893 M. leprae gene sequence:M.leprae-specific (# 114) u1935i 22673-21702 M. leprae gene sequence:M.leprae-specific (# 115) u1935j 25445-24921 M. leprae gene sequence:M.leprae-specific (# 116) u1935k 34517-34029 M. leprae gene sequence:M.leprae-specific (# 117)

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a cold-shock protein(CSPA). The protein, CSPA, is involved in cellular regulation processes.The nucleotides encoding CSPA are 27760 to 28176 of Seq. ID No. 26. Theputative amino acid sequence is set forth in Seq. ID No. 102.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a DNA helicase similarto a human DNA excision repair protein. The enzyme is involved in DNAsynthesis and replication. The nucleotides encoding this helicase are20362 to 19211 of Seq. ID No. 26. The putative amino acid sequence isset forth in Seq. ID No. 103.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding the M. leprae fattyacid oxidation complex beta subunit beta-ketothiolase (FadA). Theenzyme, FadA is involved in intermediary metabolism. The nucleotidesFadA are 7672 to 8880 of Seq. ID No. 26. The putative amino acidsequence is set forth in Seq. ID No. 104.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding the multifunctionalfatty acid oxidation complex alpha subunit (FadB). The enzyme, FadB, isinvolved in intermediary metabolism. The nucleotides encoding FadB are8855 to 11029 of Seq. ID No. 26. The putative amino acid sequence is setforth in Seq. ID No. 105.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding the M. leprae enzymeindolepyruvate decarboxylase (DCIP). The enzyme, DCIP, is involved inintermediary metabolism. The nucleotides encoding DCIP are located atpositions 4228 to 2522 of Seq. ID No. 26. The putative amino acidsequence is set forth in Seq. ID No. 106.

One non-naturally occurring nucleic acid of the present invention has asequence which corresponds to a sequence encoding a putative calciumbinding protein. The protein is involved in intermediary metabolism. Thenucleotides encoding this protein are 37098 to 36712 of Seq. ID No. 26.The putative amino acid sequence is set forth in Seq. ID No. 107.

Non-naturally occurring nucleic acids having a sequence of twenty ormore nucleotides which correspond to a sequence of Mycobacterium lepraeare useful as probes to identify Mycobacterium leprae genes orhomologous genes in other mycobacteria or other related bacterialspecies. Nucleic acid sequences that are specific to Mycobacteriumleprae genes include nucleotides at positions 4745 to 5074, nucleotides35805 to 34525, nucleotides 16602 to 17078, nucleotides 36372 to 37181,nucleotides 6871 to 6071, nucleotides 20856 to 20293, nucleotides 21696to 20893, nucleotides 22673 to 21702, nucleotides 25445 to 24921, andnucleotides 34517 to 34029 of Seq. ID No. 26. The putative amino acidsequences are set forth in Seq. ID Nos. 108-117.

One embodiment of the present invention features a non-naturallyoccurring protein or peptide of Mycobacterium tuberculosis. The proteinor peptide preferably corresponds to a sequence encoded by Seq. IDNo. 1. Preferably the protein or peptide is an antigen, or is involvedin antibiotic or cell wall synthesis, intermediary metabolism, transportprocesses, nucleic acid biosynthesis or modification, cell division orantibiotic/drug resistance. Proteins and peptides of Mycobacteriumtuberculosis are exemplified by Seq. ID Nos. 2-22, 118 and 119.

A further embodiment features a non-naturally occurring protein orpeptide of Mycobacterium leprae. The protein or peptide preferablycorresponds to a sequence within Seq. ID Nos. 23-26 or 120-140.Preferably, the protein or peptide is an antigen, or a protein involvedin antibiotic or cell wall synthesis, intermediary metabolism, transportprocesses, nucleic acid biosynthesis or modification, cell division orantibiotic/drug resistance. Proteins and peptides of Mycobacteriumleprae are exemplified by Seq. ID Nos. 27-117 and 141-411.

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.120 are summarized in Table VI set forth below.

TABLE VI Gene Position Enzyme name Function drrA 25472-26569daunorubicin :transport resistance protein drrB 26533-27432transmembrane :transport ATPase drrC 27423-28163 probable membrane:transport component pksA  35-1963 polyketide synthase:synth:lipid:antibiotic pksB 1501-4395 polyketide synthase:synth:lipid:antibiotic pksC 4396-8736 polyketide synthase:synth:lipid:antibiotic pksD  8971-15621 polyketide synthase:synth:lipid:antibiotic pksE 15573-21065 polyketide synthase:synth:lipid:antibiotic pksF 21088-25563 polyketide synthase:synth:lipid:antibiotic u1518a 28289-29578 match to M. bovis :M.leprae-specific MAS orf4 u1518b 32481-33395 M. leprae gene :M.leprae-specific sequence u1518c 30755-30982 M. leprae gene :M.leprae-specific sequence

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptide within Seq. ID No. 121are summarized in Table VII set forth below.

TABLE VII Gene Position Enzyme name Function acd 24367-25548 acyl-coAdehydrogenase :metab bccA 16077-14224 biotin carboxyl carrier :metabprotein d1308a 7737-7183 probable dehydrogenase :metab d1308b 8117-7905aldehyde dehydrogenase :metab latB 10602-10970 lysine 6-amino :synth:aatransferase latB 11263-11829 lysine 6-amino :synth:aa transferase pabB28229-27210 probable antigen B :antigen precursor pccB 20220-18535propionyl-coA :metab carboxylase pur6 23643-24284 phosphoribosylamino-:catab:tca imidazole carbox.. purK 22402-23769 phosphoribosylamino- :M.leprae-specific imidazole carbox . . . rpsB 13687-14100 sigma factor B:regulation thtR 17448-16555 thiosulfate :metab sulfotransferase t1308a28904-28638 transport protein :transport u1308a 4339-4593 M. leprae genesequence :M. leprae-specific u1308b 4822-5190 M. leprae gene sequence:M. leprae-specific u1308c 4385-4654 M. leprae gene sequence :M.leprae-specific u1308d 32525-33139 M. leprae gene sequence :M.leprae-specific u1308e 13080-13358 M. leprae gene sequence :M.leprae-specific u1308f 20175-20507 M. leprae gene sequence :M.leprae-specific u1308g 25497-25760 M. leprae gene sequence :M.leprae-specific u1308h 29565-30092 M. leprae gene sequence :M.leprae-specific u1308i 3819-3325 M. leprae gene sequence :M.leprae-specific u1308j 9309-9025 M. leprae gene sequence :M.leprae-specific u1308k 22368-21676 M. leprae gene sequence :M.leprae-specific u1308l 22368-21676 M. leprae gene sequence :M.leprae-specific u1308m 30058-29828 M. leprae gene sequence :M.leprae-specific u1308n 33166-32813 M. leprae gene sequence :M.leprae-specific u1308o 32734-32330 M. leprae gene sequence :M.leprae-specific u1308p 1121-2224 M. leprae gene sequence :M.leprae-specific u1308q 21071-21721 M. leprae gene sequence :M.leprae-specific u1308r 18512-17646 match to 21.2 kDa :M. leprae-specificprotein (yhde_ecoli) u1308s 7193-6450 M. leprae gene sequence :M.leprae-specific

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.122 are summarized in Table VIII set forth below.

TABLE VIII Gene Position Enzyme name or activity Function bioA39641-38292 adenosylmethionine- :synth:cofac:biotin 8-amino-7-oxon . . .bioB 35815-34661 biotin synthetase :synth:cofac:biotin bioD 37191-36451dethiobiotin synthase :synth:cofac:biotin bioF 38291-37128 8-amino-7-:synth:cofac:biotin oxononanoate synthase echa 1673-771  enoyl-CoAhydratase :catab:lipid:betaox ligA  9978-10628 coenzyme A ligase :M.leprae-specific m1170a 23927-17547 mycocerosic acid :synth:lipidsynthase m1170b 7464-7087 polyketide synthase :synth:lipid m1170c5853-5548 polyketide synthase :synth:lipid nadA 31541-30432 quinolinatesynthase :synth:cofac:nad nadB 30657-29158 L-aspartate oxidase:synth:cofac:nad nadC 28896-27994 nicotinate-nucleotide :synth:cofac:nadpyrophosphorylase r1170a 2601-2861 resistance protein :resistance t1170a16050-13018 transport protein :transport u1170a 9168-8884 MycobacteriumMCAS- :M. leprae-specific associated gene u1170b 17493-16051 weak matchto surfactin :M. leprae-specific synthase u1170c 4045-3581 weak match tochalcone :M. leprae-specific synthases u1170d  8875-10011MCAS-associated protein :M. leprae-specific U1170e 40416-41171 p60homolog of listeria :M. leprae-specific invasion protein u1170f4507-4767 M. leprae gene sequence :M. leprae-specific U1170g 31411-32289M. leprae gene sequence :M. leprae-specific U1170h 33070-33471 M. lepraegene sequence :M. leprae-specific u1170i 39532-39804 M. leprae genesequence :M. leprae-specific u1170j 2093-2725 M. leprae gene sequence:M. leprae-specific u1170k 10598-11350 M. leprae gene sequence :M.leprae-specific u1170l 2916-3173 M. leprae gene sequence :M.leprae-specific u1170m 11403-12590 M. leprae gene sequence :M.leprae-specific u1170n 29546-28851 M. leprae gene sequence :M.leprae-specific u1170o 34561-33800 M. leprae gene sequence :M.leprae-specific u1170p 36171-35953 M. leprae gene sequence :M.leprae-specific

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.123 are summarized in Table IX set forth below.

TABLE IX Gene Position Enzyme name or activity Function adaB 30510-31031adenosine deaminase :repair:metab ars 7367-6513 arylsulfatase :catabc1549a 13733-13236 k⁺channel protein :transport cysM 9487-9263 cysteinesynthase B :synth:aa glbA 20087-20614 1,4-alpha-glucan :synth:carbobranching enzyme glbB 21425-21721 1,4-alpha-glucan :synth:carbobranching enzyme glbC 21722-22156 1,4-alpha-glucan :synth:carbobranching enzyme glbD 20676-20894 1,4-alpha-glucan :synth:carbobranching enzyme glbE 21039-21509 1,4-alpha-glucan :synth:carbobranching enzyme glr 8234-7368 glutamate racemase :synth:aa rnpH6432-5644 ribonuclease PH :translation:tRNA rrnF  5S rRNA ribosomal RNA:translation:rRNA rrnL 35S rRNA ribosomal RNA :translation:rRNA rrnS 16SrRNA ribosomal RNA :translation:rRNA rx1549a 2253-2029 acetoacetyl-CoA:redox reductase t1549a 19490-19170 acetyltransferase :synth thi123184-22417 thioredoxin :redox thiL 24489-23299 acetyl-coa :synth:PHBacetyltransferase u1549a 11993-10752 nylonase-like :M. leprae-specificu1549b 5852-4947 M. leprae gene sequence :M. leprae-specific u1549c17362-16871 M. leprae gene sequence :M. leprae-specific u1549d15871-15551 M. leprae gene sequence :M. leprae-specific u1549e 9262-8945M. leprae gene sequence :M. leprae-specific u1549f 8944-8183 M. lepraegene sequence :M. leprae-specific u1549g 12574-11840 M. leprae genesequence :M. leprae-specific u1549h 1756-1974 M. leprae gene sequence:M. leprae-specific u1549i 32119-32415 M. leprae gene sequence :M.leprae-specific u1549j 4535-4906 M. leprae gene sequence :M.leprae-specific u1549k 24494-25039 M. leprae gene sequence :M.leprae-specific u1549l 35735-36052 M. leprae gene sequence :M.leprae-specific u1549m 36071-36361 M. leprae gene sequence :M.leprae-specific u1549n 4155-4550 M. leprae gene sequence :M.leprae-specific u1549o 19122-19421 M. leprae gene sequence :M.leprae-specific u1549p 27441-27722 M. leprae gene sequence :M.leprae-specific u1549q 30279-30494 M. leprae gene sequence :M.leprae-specific u1549r 32346-32780 M. leprae gene sequence :M.leprae-specific u1549s 28692-28183 M. leprae gene sequence :M.leprae-specific u1549t 15489-15094 M. leprae gene sequence :M.leprae-specific u1549u 1515-1138 M. leprae gene sequence :M.leprae-specific u1549v 18653-18051 M. leprae gene sequence :M.leprae-specific u1549w 16790-16266 M. leprae gene sequence :M.leprae-specific u1549x 12866-12453 M. leprae gene sequence :M.leprae-specific u1549y 29230-28556 M. leprae gene sequence :M.leprae-specific u1549z 26107-25427 M. leprae gene sequence :M.leprae-specific

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.124 are summarized in Table X set forth below.

TABLE X Gene Position Enzyme name Function d2235a  977-1282dehydrogenase :metab d2335b 1500-1901 dehydrogenase :metab dapF19144-18239 diaminopimelate :synth:aa epimerase hflX 18329-16710 hflxprotein E. coli :M. leprae-specific lexA 7697-8425 lexA:regulatory:repair miaA 20197-19196 tRNA:isopentenyltransferase:translation pgsA 35747-35088 cdp-diacylglycerol- :synth:lipidglycerol-3-phosp recA 30502-28253 recA :recombination thyA 5415-5711thymidylate synthase :synth:nt tra9 14824-15039 transposase :transposonu2235a 6806-6318 match to ybaD_Ecoli :M. leprae-specific u2235b28290-27772 recA-related ORF :M. leprae-specific u2235c 31946-30756 crtXUDP- :M. leprae-specific glucuronosyl- transferase u2235d 7489-6944 M.leprae gene :M. leprae-specific sequence u2235e 20835-20128 M. lepraegene :M. leprae-specific sequence u2235f 26340-25534 M. leprae gene :M.leprae-specific sequence u2235g 27755-26160 M. leprae gene :M.leprae-specific sequence u2235h 33858-33313 M. leprae gene :M.leprae-specific sequence u2235i 10587-8464  Corynebacterium M. :M.leprae-specific leprae-specific u2235j 2896-3927 M. leprae gene :M.leprae-specific sequence u2235k 34556-34050 M. leprae gene :M.leprae-specific sequence u2235l 34222-33722 M. leprae gene :M.leprae-specific sequence u2235m 35718-36032 M. leprae gene :M.leprae-specific sequence u2235n 3937-4191 weak match to :M.leprae-specific haloacetate dehalogenase u2235o 5125-5343 M. leprae gene:M. leprae-specific sequence u2235p 11383-11604 M. leprae gene :M.leprae-specific sequence u2235q 24715-25299 M. leprae gene :M.leprae-specific sequence u2235r 32479-32832 M. leprae gene :M.leprae-specific sequence u2335s 1283-1714 M. leprae gene :M.leprae-specific sequence u2235t 4970-5296 M. leprae gene :M.leprae-specific sequence u2235u 12323-12664 M. leprae gene :M.leprae-specific sequence u2235v 22556-22801 M. leprae gene :M.leprae-specific sequence u2235w 21376-21143 M. leprae gene :M.leprae-specific sequence u2235x 30885-32420 M. leprae gene :M.leprae-specific sequence u2235y 33312-33082 M. leprae gene :M.leprae-specific sequence u2235z 16431-16171 M. leprae gene :M.leprae-specific sequence u2235aa 15771-15535 M. leprae gene :M.leprae-specific sequence u2235bb 2583-2272 M. leprae gene :M.leprae-specific sequence u2235cc 16091-15858 M. leprae gene :M.leprae-specific sequence u2335dd 15177-15389 M. leprae gene :M.leprae-specific sequence u2235ee 30751-30539 M. leprae gene :M.leprae-specific sequence

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.125 are summarized in Table XI set forth below.

TABLE XI Enzyme name Gene Position or activity Function apt 9663-9400adenine :DNA phosphoribosyl- transferase apt2 9538-9005 adenine :ntphosphoribosyl- transferase cypH 5736-6650 proline cis-trans :M.leprae-specific isomerase-like ligA 16651-19251 long-chain fatty:lipid:metab acid-CoA ligase gabT 15206-16630 aminotransferase :M.leprae-specific pol1 19055-20293 polyketide synthase :synth:lipid[Anabaena sp.] ruvA 22895-22275 DNA repair protein :repair ruvB22408-21230 DNA repair protein :repair ruvC 23488-22883 DNA repairprotein :repair secD 14403-12196 protein secretion :transport secF12706-11429 protein secretion :transport spoT 9168-6709 guanosine 3′ 5′:synth:nt bis (diphosphate) 3′ . . . syH 4942-3656 histidinyl-tRNA:synth:protein synthetase yebC 28331-27555 yebC Ecoli :M.leprae-specific homolog u1177a 23715-23464 M. leprae gene :M.leprae-specific sequence u1177b 10824-9718  dciAE gene :M.leprae-specific product Bsubtilus u1177c 37821-38612 ycb9_ :M.leprae-specific Psede/ycfA_(—) Ecoli homolog u1177f 25360-25803 M.leprae gene :M. leprae-specific sequence u1177g 7681-8391 Ecoli 77.2 Kd:M. leprae-specific protein homolog u1177h 36689-36009 weak match to :M.leprae-specific gramicidin synth u1177i 26410-26790 M. leprae gene :M.leprae-specific sequence u1177j 26923-27207 M. leprae gene :M.leprae-specific sequence u1177k 33331-33618 M. leprae gene :M.leprae-specific sequence u1177l 35257-35559 M. leprae gene :M.leprae-specific sequence u1177m 20321-20662 M. leprae gene :M.leprae-specific sequence u1177n 28658-28897 M. leprae gene :M.leprae-specific sequence u1177o 24507-25343 M. leprae gene :M.leprae-specific sequence u1177p 32772-33068 M. leprae gene :M.leprae-specific sequence u1177q 39762-39442 M. leprae gene :M.leprae-specific sequence u1177r 31446-31000 M. leprae gene :M.leprae-specific sequence u1177s 30642-30397 M. leprae gene :M.leprae-specific sequence u1177t 30989-30717 M. leprae gene :M.leprae-specific sequence u1177u 21368-21078 M. leprae gene :M.leprae-specific sequence u1177v 37342-37052 M. leprae gene :M.leprae-specific sequence u1177w 15028-14678 M. leprae gene :M.leprae-specific sequence u1177x 6268-5732 M. leprae gene :M.leprae-specific sequence u1177y 29259-28531 S. coelicolor :synth:aahistidine biosynthesis u1177aa 3393-3073 ipiB1 gene product :M.leprae-specific [Phytophthora sp] u1177ab 40088-39681 dedA protein Ecoli:M. leprae-specific u1177ac 11570-10143 M. leprae gene :M.leprae-specific sequence u1177ad 10447-10235 M. leprae gene :M.leprae-specific sequence u1177ae 5698-4943 M. leprae gene :M.leprae-specific sequence

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.126 are summarized in Table XII set forth below.

TABLE XII Gene Position Enzyme name Function a2126a 16422-14530 ATPase:M. leprae-specific bacA 38533-38192 bacitracin resistance prot:resistance homology Ecoli cysQ 35494-34562 match to E. coli cycQ :M.leprae- specific his1 22371-21487 ATP-phosphoribosyl :synth:aatransferase his2 22661-22353 phosphoribosyl-amp :synth:aa cyclohydrolaselysP 28847-29650 lysine-specific permease :transport Ecoli metH25145-24129 methionine synthase :synth:aa metH 27780-25078 methioninesynthase :synth:aa aroP 29574-30434 aromatic aa transport :transportEcoli aroP 30482-32080 aromatic aa transport :transport Ecoli pimT18549-17647 protein-beta-aspartate :catab:prot methylt-ase prcB8970-8047 proteasome, beta subunit :catab:prot pyrD 40218-41330dihydroorotate :synth:nt dehydrogenase syC 34469-33606 cys tRNAsynthetase :translation u2126a 1095-133  match to yigU and yigV :M.leprae- E. coli specific u2126b 1470-1132 match to yigT :M. leprae- E.coli specific u2126c 13279-13665 M. leprae gene sequence :M. leprae-specific u2126d 14335-14763 M. leprae gene sequence :M. leprae- specificu2126e 22993-23226 M. leprae gene sequence :M. leprae- specific u2126f27793-28779 M. leprae gene sequence :M. leprae- specific u2126g40207-40503 M. leprae gene sequence :M. leprae- specific u2126h21422-21694 M. leprae gene sequence :M. leprae- specific u2126i39908-40240 M. leprae gene sequence :M. leprae- specific u2126j17189-17533 Match to Bsu ORF :M. leprae- specific u2126k 41405-41887 M.leprae gene sequence :M. leprae- specific u2126l 16641-17231 M. lepraegene sequence :M. leprae- specific u2126m 20814-21263 M. leprae genesequence :M. leprae- specific u2126n 23049-23768 M. leprae gene sequence:M. leprae- specific u2126o 38937-39989 M. leprae gene sequence :M.leprae- specific u2126p 37821-36883 M. leprae gene sequence :M. leprae-specific u2126q 36390-35416 M. leprae gene sequence :M. leprae- specificu2126r 20103-19813 M. leprae gene sequence :M. leprae- specific u2126s12489-11986 M. leprae gene sequence :M. leprae- specific u2126t10764-9142 M. leprae gene sequence :M. leprae- specific u2126u 1776-1471M. leprae gene sequence :M. leprae- specific u2126v 18554-19447mycobacteriophage L5 :M. leprae- homology specific u2126w 38246-37632 M.leprae gene sequence :M. leprae- specific u2126x 7187-6822 M. lepraegene sequence :M. leprae- specific u2126y 5618-5373 M. leprae genesequence :M. leprae- specific u2126z 4916-3861 M. leprae gene sequence:M. leprae- specific u2126aa 2522-1701 M. leprae gene sequence :M.leprae- specific u2126ab 36910-36215 M. leprae gene sequence :M. leprae-specific u2126ac 33577-33215 M. leprae gene sequence :M. leprae-specific u2126ad 19744-19427 M. leprae gene sequence :M. leprae-specific u2126ae 17800-17372 M. leprae gene sequence :M. leprae-specific u2212af 13921-13640 M. leprae gene sequence :M. leprae-specific u2126ag 13345-12950 M. leprae gene sequence :M. leprae-specific u2126ah 8053-7253 M. leprae gene sequence :M. leprae- specificu2126ai 6868-6569 M. leprae gene sequence :M. leprae- specific u2126aj4114-3443 M. leprae gene sequence :M. leprae- specific u2126ak 3442-2441M. leprae gene sequence :M. leprae- specific u2126al 23716-23928 ham34gene product :M. leprae- [Bremia lactucae] specific

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.127 are summarized in Table XIII set forth below.

TABLE XIII Gene Position Enzyme name or activity Function capP26821-24005 phosphoenolpyruvate :catab:tca carboxylase coxX 17264-16155cytochrome assembly :redox factor g3p 32185-31115 glyceraldehyde-3-:catab:carbo:glyco phosphate deHase g6pdA 21301-21672glucose-6-phosphate :M. leprae-specific dehydrogenase g6pdB 20997-21308glucose-6-phosphate :M. leprae-specific dehydrogenase g6pdC 21633-21884glucose-6-phosphate :M. leprae-specific dehydrogenase nifS 3821-2514homologue of nitrogen :M. leprae-specific fixation gene nifU 2571-2020homologue of nitrogen :M. leprae-specific fixation gene pgk 31145-29862phosphoglycerate kinase :catab:carbo:glyco t1496a 4693-3773 trafficATPase :transport t1496b 11040-11993 drrA traffic ATPase :transport tkt17317-19419 transketolase :synth:carbo tpiS 29886-29080 TPI (TIM):catab:carbo:glyco u1496a 15109-14645 M. leprae gene sequence :M.leprae-specific u1496b 718-275 M. leprae gene sequence :M.leprae-specific u1496c  9253-11049 M. leprae gene sequence :M.leprae-specific u1496d 15082-15306 M. leprae gene sequence :M.leprae-specific u1496e 15499-15978 M. leprae gene sequence :M.leprae-specific u1496f 19885-20562 M. leprae gene sequence :M.leprae-specific u1496g 22186-23022 M. leprae gene sequence :M.leprae-specific u1496h 23023-23781 match to yeast ORF :M.leprae-specific u1496i  962-1405 M. leprae gene sequence :M.leprae-specific u1496j 11762-12790 M. leprae gene sequence :M.leprae-specific u1496k 13397-14356 M. leprae gene sequence :M.leprae-specific u1496l 19085-19915 transaldolase :M. leprae-specificu1496m 32324-33412 M. leprae gene sequence :M. leprae-specific u1496n28857-28507 M. leprae gene sequence :M. leprae-specific u1496o27336-27085 M. leprae gene sequence :M. leprae-specific u1496p24039-23698 M. leprae gene sequence :M. leprae-specific u1496q 5844-4552yca_yeast homolog :M. leprae-specific u1496r 1728-1459 fungus ORFhomolog :M. leprae-specific u1496s 29207-28821 M. leprae gene sequence:M. leprae-specific u1496t 28358-28131 M. leprae gene sequence :M.leprae-specific u1496u 22439-22086 M. leprae gene sequence :M.leprae-specific u1496v 21134-20700 M. leprae gene sequence :M.leprae-specific u1496w 9248-8388 M. leprae gene sequence :M.leprae-specific u1496x 8387-5727 yca3_yeast homolog :M. leprae-specificu1496y 2087-1698 M. leprae gene sequence :M. leprae-specific u1496z27079-26840 M. leprae gene sequence :M. leprae-specific

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.128 are summarized in Table XIV set forth below.

TABLE XIV Gene Position Enzyme name Function cfa 22851-23921cyclopropane-fatty- :synth:lipid acyl-phospholip Ecoli cysG 18016-16592uroporphyrin-iii :synth:cofac:heme c-methylt-ase gsa 7593-6190glutamate-1- :M. leprae-specific semialdehyde 2,1-aminot-ase hem120446-18974 glutamyl-trna :synth:cofac:heme reductase hem2 16297-15284delta-aminolevulinic :synth:cofac:heme acid deh2oase hem3 18490-18017porphobilinogen :synth:cofac:heme deaminase hem3 18969-18427porphobilinogen :synth:cofac:heme deaminase phoP 36607-35969 phosphatesensor :regulatory phoR 37960-36608 phosphate sensor :regulatory pmgY38851-38096 phosphoglycerate :catab:carbo:glyco mutase ppx 31790-30552exopolyphosphatase :catab proC 27878-26970 pyrroline-5- :synth:aacarboxylate reductase serB 21424-22380 phosphoserine :synth:aaphosphatase t2168a 10404-10156 permease :transport t2168b 11030-10557permease :transport u2168a 31534-32508 M. leprae gene :M.leprae-specific sequence u2168b 26163-25567 match to rfbE :M.leprae-specific u2168c 5624-4917 weak match to :M. leprae-specificthioredoxin u2168d 26844-26569 weak match to :M. leprae-specificexcisionase u2168e 39445-38852 M. leprae gene :M. leprae-specificsequence u2168f 40823-39480 match to rfaG :synth or susY u2168g42424-42741 M. leprae gene :M. leprae-specific sequence u2168h 4965-4132M. leprae gene :M. leprae-specific sequence u2168i 8633-8926 M. lepraegene :M. leprae-specific sequence u2168j 22442-22984 M. leprae gene :M.leprae-specific sequence u2168k 33293-33805 M. leprae gene :M.leprae-specific sequence u2168l 34346-34630 M. leprae gene :M.leprae-specific sequence u2168m 34827-35060 M. leprae gene :M.leprae-specific sequence u2168n 35061-35300 M. leprae gene :M.leprae-specific sequence u2168o 35397-35762 M. leprae gene :M.leprae-specific sequence u2168p 25068-23950 M. leprae gene :M.leprae-specific sequence u2168q 15246-14935 M. leprae gene :M.leprae-specific sequence u2168r 13170-12910 M. leprae gene :M.leprae-specific sequence u2168s 12411-12139 M. leprae gene :M.leprae-specific sequence u2168t 12009-11608 M. leprae gene :M.leprae-specific sequence u2168u 3177-2509 M. leprae gene :M.leprae-specific sequence u2168v 591-247 M. leprae gene :M.leprae-specific sequence u2168w 33014-32673 M. leprae gene :M.leprae-specific sequence u2168x 30281-29487 M. leprae gene :M.leprae-specific sequence u2168y 29486-28614 M. leprae gene :M.leprae-specific sequence u2168z 25703-25059 M. leprae gene :M.leprae-specific sequence u2168aa 4145-2997 M. leprae gene :M.leprae-specific sequence u2168ab 6244-5582 M. leprae gene :M.leprae-specific sequence u2168ac 30576-30199 M. leprae gene :M.leprae-specific sequence u2168ad 26443-26225 M. leprae gene :M.leprae-specific sequence u2168ae 16682-16320 M. leprae gene :M.leprae-specific sequence u2168af 2557-1529 M. leprae gene :M.leprae-specific sequence u2168ag 11776-11420 match to :catabphospholipase u2168ah 41938-42423 M. leprae gene :M. leprae-specificsequence

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.129 are summarized in Table XV set forth below.

TABLE XV Enzyme name Gene Position or activity Function abc1 40693-39500Yeast mt protein :transport cysA 17806-18105 sulfate permease :transportcysW 17098-17553 sulfate permease :transport dnaJ 24656-25825 heat shock:stress erA 30210 31199 GTPase? :M. leprae-specific lepA  9501-11474lethal when :M. leprae-specific overexpressed phoH 26887-27978 phosphatemetabolism :metab recQ 37286-38086 DNA helicase RECQ :repair subI15124-16245 Sulfate-binding protein :transport uvrD 35866-37278 DNAhelicase :repair ygrp 23572-24180 match to 38.8 kD :M. leprae-specificprotein (SP:P30727) u1937a 1195-1440 M. leprae gene :M. leprae-specificsequence u1937b 3361-5052 match to 35.9 kDa :M. leprae-specific protein(PIR:JQ1236) u1937c 27994-28545 M. leprae gene :M. leprae-specificsequence u1937d 28972-29418 M. leprae gene :M. leprae-specific sequenceu1937e 32542-33534 M. leprae gene :M. leprae-specific sequence u1937f1481-1723 M. leprae gene :M. leprae-specific sequence u1937g 1730-2935M. leprae gene :M. leprae-spedfic sequence u1937h 5903-6868 M. lepraegene :M. leprae-specific sequence u1937i 6881-7465 M. leprae gene :M.leprae-specific sequence u1937j 8642-8857 M. leprae gene :M.leprae-specific sequence u1937k 33431-34417 match to (gp:z26494) :M.leprae-spedfic u1937m 14585-14881 M. leprae gene :M. leprae-specificsequence u1937n 16523-16933 sulfate permease :M. leprae-specific Tprotein u1937o 26192-26629 M. leprae gene :M. leprae-specific sequenceu1937p 38468-38806 M. leprae gene :M. leprae-specific sequence u1937q 750-1076 M. leprae gene :M. leprae-specific sequence u1937r 4923-6029M. leprae gene :M. leprae-speciflc sequence u1937s 19449-19667 M. lepraegene :M. leprae-specific sequence u1937t 20292-20852 M. leprae gene :M.leprae-specific sequence u1937u 24126-24617 M. leprae gene :M.leprae-specific sequence u1937v 25728-26255 match to S.coelicolor :M.leprae-specific gene u1937w 28677-29045 M. leprae gene :M.leprae-specific sequence u1937x 29835-30173 M. leprae gene :M.leprae-specific sequence u1937y 38112-38543 M. leprae gene :M.leprae-specific sequence u1937z 18537-18292 M. leprae gene :M.leprae-specific sequence u1937aa 8397-7645 M. leprae gene :M.leprae-specific sequence u1937ab 39581-38883 M. leprae gene :M.leprae-specific sequence u1937ac 21140-20898 M. leprae gene :M.leprae-specific sequence u1937ad 19160-18858 M. leprae gene :M.leprae-specific sequence u1937ae 18317-17802 M. leprae gene :M.leprae-specific sequence u1937af 11795-11532 M. leprae gene :M.leprae-specific sequence u1937ag 42766-41321 M. leprae gene :M.leprae-specific sequence u1937ah 38932-38603 M. leprae gene :M.leprae-specific sequence u1937aj 35383-35072 M. leprae gene :M.leprae-specific sequence u1937ak 13666-13298 M. leprae gene :M.leprae-specific sequence u1937al 3235-2972 M. leprae gene :M.leprae-specific sequence

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.130 are summarized in Table XVI set forth below.

TABLE XVI Enzyme name Gene Position or activity Function acvS32477-32112 cysteinyl synthetase :synth:aa alr 9181-7928 alanineracemase :synth:wall chA 4066-3749 10 kd chaperonin :antigen:stress dceA10449-10117 glutamate :metab decarboxylase(DCEA) dceB 10104-9784 glutamate :metab decarboxylase(DCEA) dceC 9977-9624 glutamate :metabdecarboxylase(DCEA) glmS 14311-12359 glucosamine-fructose- :M.leprae-specific 5-phosphate groE1 3679-2057 groE1 protein-m.:antigen:stress leprae ilvi1 29556-28255 acetolactate synthase:synth:aa:iv:1 ilvi2 29864-29337 acetolactate synthase :synth:aa:iv:1rim 6561-5383 ribosomal :translation acetyltransferase rl13 20260-19787large ribosomal :translation subunit p13 rs9 19886-19329 small ribosomal:translation subunit p9 u0229a 1054-1458 Regulatory protein :regulationu0229b 14239-15270 M. leprae gene :M. leprae-specific sequence u0229c24448-24750 M. leprae gene :M. leprae-specific sequence u0229d26443-26712 M. leprae gene :M. leprae-specific sequence u0229e35122-35457 M. leprae gene :M. leprae-specific sequence u0229f13748-14269 M. leprae gene :M. leprae-specific sequence u0229g15620-16030 M. leprae gene :M. leprae-specific sequence u0229h 9024-9443M. leprae gene :M. leprae-specific sequence u0229i 25887-26549 M. lepraegene :M. leprae-specific sequence u0229j 27486-27274 pyrophosphokinase:M. leprae-specific u0229k 21717-21499 M. leprae gene :M.leprae-specific sequence u0229l 20796-20482 M. leprae gene :M.leprae-specific sequence u0229m 17346-17002 M. leprae gene :M.leprae-specific sequence u0229n 15735-15499 M. leprae gene :M.leprae-specific sequence u0229o 8088-6937 M. leprae gene :M.leprae-specific sequence u0229p 825-1  M. leprae gene :M.leprae-specific sequence u0229q 31751-31473 M. leprae gene :M.leprae-specific sequence u0229r 31118-30891 M. leprae gene :M.leprae-specific sequence u0229s 30179-29865 M. leprae gene :M.leprae-specific sequence u0229t 23951-23682 M. leprae gene :M.leprae-specific sequence u0229u 1076-810  M. leprae gene :M.leprae-specific sequence u0229v 24568-24311 M. leprae gene :M.leprae-specific sequence u0229w 23686-23450 M. leprae gene :M.leprae-specific sequence u0229x 22795-22484 M. leprae gene :M.leprae-specific sequence u0229y 12019-11570 M. leprae gene :M.leprae-specific sequence u0229z 18863-17832 match to 47.5 kd :M.leprae-specific E. coli protein u0229aa 19279-18701 match to 47.5 kd :M.leprae-specific E. coli protein u0229ab 5530-4514 match to 36.0 kd :M.leprae-specific E. coli ribosomal u0229ac 7058-6459 match to 16.9 kd :M.leprae-specific E. coli protein u0229ad 11891-10677 match to E. coliamiB :M. leprae-specific 5′ regulatory

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.131 are summarized in Table XVII set forth below.

TABLE XVII Enzyme name Gene Position or activity Function ag42 5160-391045 KDa antigen :antigen (gp:z21952 chA 40918-40409 10 KDa chaperonin:antigen:stress choD 30517-28730 cholesterol oxidase :catab:lipid groE140339-38717 groE1 :antigen:stress guaA 23093-21258 gmp synthase:synth:nt impA 31856-30504 inosine-5′- :synth:nt monophosphate dehydrogimpB 33271-31640 inosine-5′- :synth:nt monophosphate dehydrog otsB 616-1908 trehalose- :M. leprae-specific phosphatase rbsB 16757-17830ribose binding :transport protein rbsC 18050-19951 ribose transport:transport protein s1620a 37713-38117 sigma factor :regulatory s1620b36241-35942 sigma factor :regulatory u1620a 6328-6816 transposase :M.leprae-specific u1620b 7618-7286 transposase :M. leprae-specific u1620c42190-40991 match to :M. leprae-specific glycoproteinase u1620f25213-27627 M. leprae gene :M. leprae-specific sequence u1620g20058-21176 M. leprae gene :M. leprae-specific sequence u1620h10682-9489  M. leprae gene :M. leprae-specific sequence u1620i 7738-8049M. leprae gene :M. leprae-specific sequence u1620j 2166-1690 M. lepraegene :M. leprae-specific sequence u1620k 5660-5262 weak match 32 KD :M.leprae-specific protein (sp:p16645) u1620l 9446-9111 M. leprae gene :M.leprae-specific sequence u1620m 2191-1979 weak match 42.1 KD :M.leprae-specific protein(sp:p29156) u1620n 2542-2282 weak match to :M.leprae-specific (gp:x66077) u1620o 9256-8447 M. leprae gene :M.leprae-specific sequence u1620p 13237-13515 M. leprae gene :M.leprae-specific sequence u1620q 13642-13956 M. leprae gene :M.leprae-specific sequence u1620r 12686-12976 M. leprae gene :M.leprae-specific sequence u1620s 12480-12836 weak match to :M.leprae-specific (gp:104527) u1620t 23445-23975 weak match 44.7 KD :M.leprae-specific protein(sp:p31049) u1620u 24399-25235 M. leprae gene :M.leprae-specific sequence u1620v 27945-27673 M. leprae gene :M.leprae-specific sequence u1620w 33375-33797 M. leprae gene :M.leprae-specific sequence u1620x 34396-34049 M. leprae gene :M.leprae-specific sequence u1620y 35014-34772 M. leprae gene :M.leprae-specific sequence u1620z 35428-35153 M. leprae gene :M.leprae-specific sequence u1620aa 35791-35549 M. leprae gene :M.leprae-specific sequence u1620ab 37444-36605 M. leprae gene :M.leprae-specific sequence u1620ac 37735-37469 M. leprae gene :M.leprae-specific sequence

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.132 are summarized in Table XVIII set forth below.

TABLE XVIII Gene Position Enzyme name Function add 16658-15639 adenosinedeaminase :catab:nt d0308a 2859-2626 dehydrogenase :M. leprae-specificd0308b 2017-1757 dehydrogenase :M. leprae-specific dhsA1 18908-19738succinate :catab:tca dehydrogenase dhsA2 17886-19190 succinate:catab:tca dehydrogenase dhsB 19618-20532 succinate :catab:tcadehydrogenase glpD 2478-703  aerobic glycerol-3- :catab phosphatedeh-ase idhpA 34023-33808 isocitrate :catab:tca dehydrogenase idhpB33644-33378 isocitrate :catab:tca dehydrogenase idhpC 34369-34142isocitrate :catab:tca dehydrogenase idhpB 33976-33593 isocitrate:catab:tca dehydrogenase idhpB 33535-33218 isocitrate :catab:tcadehydrogenase met2 36069-36410 homoserine :synth:aa acetyltransferasemet5a 34828-35145 homoserine synth:aa sulfhydralase met5b 35410-36102homoserine :synth:aa sulfhydralase met5c 35087-35434 homoserine:synth:aa sulfhydralase pbpB 25732-26523 penicillin binding :wallprotein pbpC 25085-25837 penicillin binding :wall protein pnpH 7955-8950purine nucleoside :catab:nt phosphorylase syW 32289-31243 Trp tRNAsynthetase :translation tpeA 32298-33221 match to :M. leprae-specifictropinesterase merA 3987-3766 match to merA :M. leprae-specific proteinu0308a 23625-23867 probable :M. leprae-specific acetyltransferase u0308b 8878-10563 match to :M. leprae-specific phosphomannomutase u0308c7438-8073 :M. leprae-specific u0308d 12202-12771 :M. leprae-specificu0308e 12202-12771 :M. leprae-specific u0308f 29947-30240 :M.leprae-specific u0308g 12926-13525 :M. leprae-specific u0308h16709-17476 :M. leprae-specific u0308i 22067-22306 :M. leprae-specificu0308j 23432-23689 :M. leprae-specific u0308k 27758-28018 :M.leprae-specific u0308l 28748-28978 :M. leprae-specific u0308m 4092-4625:M. leprae-specific u0308n 12708-13025 match to nusG protein :M.leprae-specific u0308o 26865-27119 possible glucose :M. leprae-specifictransporter u0308p 28953-28495 :M. leprae-specific u0308q 25818-25273:M. leprae-specific u0308r 14874-14437 :M. leprae-specific u0308s11262-10900 match to uracil :M. leprae-specific phosphoribosyl-transferase u0308t 5475-5038 :M. leprae-specific u0308u 35264-34986 :M.leprae-specific u0308v 23081-22758 :M. leprae-specific u0308w22592-22317 :M. leprae-specific u0308x 18272-17928 :M. leprae-specificu0308y 11018-10704 match to uracil :M. leprae-specific phosphoribosyl-transferase u0308z 7823-7425 :M. leprae-specific u0308aa 31480-30290match to :M. leprae-specific metallothioneins u0308ab 21010-20654 :M.leprae-specific u0308ac 6043-5720 :M. leprae-specific

Mycobacterium leprae peptides, the function of such peptides, and thenucleotide positions associated with such peptides within Seq. ID No.133 are summarized in Table XIX set forth below.

TABLE XIX Gene Position Enzyme name Function poIIA 2289-643  DNAPolymerase I- :replication M.tuberculosis poIIB 3382-2273 DNA PolymeraseI :replication pyrG 27752-25824 CTP synthase :synth:nt recN 31868-30105recombinase :recombination syy 39193-38282 Tyr tRNA-synthetase:translation u0247a 33657-32824 hemolysin :M. leprae-specific u0247b23927-22857 M. leprae-specific :M. leprae-specific family u0247c8082-7870 probable oxidase :M. leprae-specific u0247d 25243-24260 matchto integrase :M. leprae-specific u0247e 19935-18493 M. leprae gene :M.leprae-specific sequence u0247f 33076-31892 utrl Sce M. leprae - :M.leprae-specific specific u0247g 21289-20543 E. coli rpsA region :M.leprae-specific protein u0247h 3617-3390 M. lepraegene :M.leprae-specific sequence u0247i 8062-8460 M. leprae gene :M.leprae-specific sequence u0247j 36797-37066 M. leprae gene :M.leprae-specific sequence u0247k 5304-5561 M. leprae gene :M.leprae-specific sequence u0247l 2870-2490 M. leprae gene :M.leprae-specific sequence u0247m 30111-28738 M. leprae gene :M.leprae-specific sequence u0247n 28737-28309 M. leprae gene :M.leprae-specific sequence u0247o 25878-25207 M. leprae gene :M.leprae-specific sequence u0247p 22881-22054 M. leprae gene :M.leprae-specific sequence u0247q 15463-15092 M. leprae gene :M.leprae-specific sequence u0247r 36647-36378 M. leprae gene :M.leprae-specific sequence u0247s 34199-33831 M. leprae gene :M.leprae-specific sequence u0247t 28106-27753 M. leprae gene :M.leprae-specific sequence u0247u 11296-10754 M. leprae gene :M.leprae-specific sequence u0247v 15883-15497 M. leprae gene :M.leprae-specific sequence u0247w 22091-21312 M. leprae gene :M.leprae-specific sequence u0247x 20558-19875 M. leprae gene :M.leprae-specific sequence u0247y 14366-14088 M. leprae gene :M.leprae-specific sequence u0247z 10739-10425 M. leprae gene :M.leprae-specific sequence u0247aa 9839-9510 M. leprae gene :M.leprae-specific sequence u0247ab 6764-6123 M. leprae gene :M.leprae-specific sequence u0247ac 4532-4245 M. leprae gene :M.leprae-specific sequence

Mycobacterium Leprae peptides, the functions of such peptides, andnucleotide positions associated with such peptides within Seq. ID No.134 are summarized in Table XX below.

TABLE XX Name SeqID Position Enzyme or Protein Name Function atp6 14114735-13968 ATP synthase A chain :metab atpA 142 11700-10009 ATPsynthase alpha chain :metab atpB 143 9167-7611 ATP synthase beta chain:metab atpC 144 13884-13627 ATP synthase C chain :metab atpD 14513120-11747 ATP synthase delta chain :metab atpE 146 7596-7213 ATPsynthase epsilon chain :metab atpF 147 13626-13093 ATP synthase B chain:metab atpG 148 10021-9095  ATP synthase gamma chain :metab dcda 14927058-25505 diaminopimelate decarboxylase :synth:aa dhom 150 25504-24176homoserine dehydrogenase :synth:aa khse 151 23107-22082 homoserinekinase :synth:aa largs 152 29031-26920 arginyl-tRNA synthetase:translation murZ 153 5599-4302 UDP-N-acetyglucosamine 1-carboxyvinyltransferase :synth:carbo orfWT 154 5657-6256 C. freundii orfW homologue:M. leprae specifi prfA 155 18264-17668 protein synthesis release factor:translation rfl 156 19325-18240 peptide chain release factor 1:regulatory rfe 157 16655-15444 rfe protein :antigen rho 158 21836-19980transcription termination factor R :regulatory:transc r131 15919879-19415 ribosomal protein L31 :synth:prot sua5 160 17342-16674 yeastSUA5 protein :mitochondrial thrc 161 24233-23097 threonine synthase:synth:aa u471a 162 7212-6763 M. leprae gene sequence :M. lepraespecific u471b 163 6400-6624 M. leprae gene sequence :M. leprae specificu471c 164 15190-14720 M. leprae gene sequence :M. leprae specific u471c165 22118-21837 M. leprae gene sequene :M. leprae specific u471d 16617762-17376 M. leprae gene sequence :M. leprae specific

Mycobacterium Leprae peptides, the functions of such peptides, andnucleotide positions associated with such peptides within Seq. ID No.135 are summarized in Table XXI below.

TABLE XXI Name SeqID Position Enzyme or Protein Name Function cciptfd167 6705-7274 P450 cytochrome,isopentenyltransf, ferridox.:synth:antibiotic corA 168 35018-33603 magnesium and cobalt transportprotein :transport dhps 169 9042-9920 dihydropteroate synthase :synthGLGC 170 13535-14827 glucose-1-phosphate adenylyltransferase:synth:carbo htra1 171 21012-22637 heat shock protein htrA:antigen:stress malf 172 29904-30938 maltose transport inner membraneprotein :transport malg 173 30883-31791 maltose transport inner membraneprotein :transport maox 174 37206-37997 malate oxireductase(NAD) :metabmdhc 175 34923-35924 malate dehydrogenase :metab mdmc 176 19306-18650O-methyltransferase :synth:nt mg1 177 11117-11827 DNA-3-methyladenineglycosidase I :chemotaxis mrp 178 25338-24019 MRP protein :synth rpoE179 19796-20395 RNA polymerase sigma-E factor :regulatory sus1 18012702-12388 sucrose synthase 1 :synth:carbo thrb2a 181 17482-17144 TnrB2protein :resistance thrb2b 182 17938-17615 TnrB2 protein :resistanceugpC 209 31784-32974 ugpC gene product :transport u1756a 183 624-941 M.leprae gene sequence :M. leprae specifi u1756b 208 986-161 serine richantigen - M. leprae :antigen u1756c 184 1698-2003 M. leprae genesequence :M. leprae specific u1756d 185 2004-2342 M. leprae genesequence :M. leprae specific u1756e 186 2511-2870 M. leprae genesequence :M. leprae specific u1756f 187 3947-2961 M. leprae genesequence :M. leprae specific u1756g 188 3803-5032 M. leprae genesequence :M. leprae specific u1756h 189 5234-4989 M. leprae genesequence :M. leprae specific u1756i 190 5865-5461 M. leprae genesequence :M. leprae specific u1756j 191 8473-8733 M. leprae genesequence :M. leprae specific u1756k 192 8619-9041 M. leprae genesequence :M. leprae specific u1756l 193  9722-10873 M. leprae genesequence :M. leprae specific u1756m 194 10893-11252 M. leprae genesequence :M. leprae specific u1756n 195 12026-12268 M. leprae genesequence :M. leprae specific u1756o 196 13176-12772 M. leprae genesequence :M. leprae specific u1756p 197 14898-15554 M. leprae genesequence :M. leprae specific u1756q 198 15921-15505 M. leprae genesequence :M. leprae specific u1756r 199 16551-16129 M. leprae genesequence :M. leprae specific u1756s 200 17051-16577 M. leprae genesequence :M. leprae specific u1756t 201 18229-17957 M. leprae genesequence :M. leprae specific u1756u 202 19570-19944 M. leprae genesequence :M. leprae specific u1756v 203 27394-26282 M. leprae genesequence :M. leprae specific u1756w 204 28547-30938 M. leprae genesequence :M. leprae specific u1756x 205 36803-37099 M. leprae genesequence :M. leprae specific u1756y 206 37769-38674 M. leprae genesequence :M. leprae specific u1756z 207 22634-22999 M. leprae genesequence :M. leprae specific

Mycobacterium Leprae peptides, the functions of such peptides, andnucleotide positions associated with such peptides within Seq. ID No.136 are summarized in Table XXII below.

TABLE XXII Name SeqID Position Enzyme or Protein Name Function 4CL1 21033126-31948 4-Coumarate-CoA ligase :metab ag84 211 27120-26890 antigenAg84 :antigen anp4 212 31293-31631 antifreeze peptide 4 precursor:regulatory cpsA 213 20816-22444 cpsA gene product :synth:carbo DHG 21422804-22529 glucose 1-dehydrogenase :synth:carbo DHMA 215 23283-23074N-acylmannosamine 1-dehydrogenase :synth:carbo glvr1 216 35001-33718glvr-1 protein :antibiotic mtrA 217 9475-8901 phosphate regulatoryprotein(M. tuberculosis) :regulatory otsa 218 27834-29330alpha,alpha-trehalose-phosphate synthase :redox pkc1 219 6931-7422protein kinase C inhibitor 1 :regulatory pur2 220 5183-3909phosphoribosylamine-glysine ligase :synth srcO 221 18805-18602 sarcosineoxidase :catab u296a 222 315-1  M. leprae gene sequence :M. lepraespecific u296b 223 1966-1733 M. leprae gene sequence :M. leprae specificu296c 224 3256-3534 M. leprae gene sequence :M. leprae specific u296d225 5498-5184 M. leprae gene sequence :M. leprae specific u296e 2266019-5636 M. leprae gene sequence :M. leprae specific u296f 2276259-6576 M. leprae gene sequence :M. leprae specific u296g 2288070-7768 M. leprae gene sequence :M. leprae specific u296g 2298488-8727 M. leprae gene sequence :M. leprae speciflc u296h 23010580-10371 M. leprae gene sequence :M. leprae specific u296i 23111570-11280 M. leprae gene sequence :M. leprae specific u296j 23212480-11617 hypothetical 26.6K protein (M. fortuitum) :M. leprae specifiu296k 233 15238-15053 M. leprae gene sequence :M. leprae specific u296l234 16952-17338 M. leprae gene sequence :M. leprae specific u296m 23517578-17276 M. leprae gene sequence :M. leprae specinc u296n 23618592-18293 M. leprae gene sequence :M. leprae specific u296o 23720439-20239 M. leprae gene sequence :M. leprae specific u296p 23820685-20894 M. leprae gene sequence :M. leprae specific u296q 23923759-23992 M. leprae gene sequence :M. leprae specific u296r 24023878-24234 M. leprae gene sequence :M. leprae specific u296s 24126577-26338 M. leprae gene sequence :M. leprae specliic u296t 24230960-30700 M. leprae gene sequence :M. leprae specific u296u 24333751-33431 M. leprae gene sequence :M. leprae specific u296v 24433437-33099 M. leprae gene sequence (ATP site) :M. leprae specific u296w245 35512-35060 M. leprae gene sequence :M. leprae specific

Mycobacterium Leprae peptides, the functions of such peptides, andnucleotide positions associated with such peptides within Seq. ID No.137 are summarized in Table XXIII below.

TABLE XXIII Name SeqID Position Enzyme or Protein Name Function ampL 246 675-2018 vacular aminopeptidase I precusor :metab:protein glnR 24722621-22818 GlnR protein (response regulator; similar to phoP):regulatory gocp 248 7775-6135 capsule gene complex(galE)(rfbB)(rfbC/D):synth:carbo icc 249 5243-6220 ICC protein :regulatory ntrB 25031848-30655 ntrB gene :regulatory pdp 251 23955-25253phosp-repressible,periplasmicphosph.-binding prot :transport phoB 25228555-27839 phosphate regulatory protein :transport pstA 253 26324-26926phosphate transport protein PSTA :transport pstB 254 26991-27812phosphate transport protein PSTB :transport pstC 255 25357-25863phosphate transport protein :transport pur1 256  9799-11472aminophosphoribosyl transferase precursor :synth:nt pur5 257 11547-12140phosphoribosylformulglycinamidine cyclo-ligase :synth:nt purL 2582834-5134 Phosphoribosylformylglycinamidine :synth:nt purM 25912023-12664 phosphoribosylamine-glycine ligase :synth:nt stad 26032890-31853 [acyl-carrier protein] desaturase precusorR :synth:lipidthi1 261 21277-20753 thioredoxin :redox thtr 262 20164-19190 thiosulfatesulfurtransferase :synth:aa u2266a 263 286-81  M. leprae gene sequence:M. leprae specific u2266b 264 512-309 M. leprae gene sequence :M.leprae specific u2266c 265 793-332 M. leprae gene sequence :M. lepraeSpecific u2266d 266 8748-8509 M. leprae gene sequence :M. lepraespecific u2266e 267 8960-8721 M. leprae gene sequence :M. lepraespecific u2266f 268 14252-13113 M. leprae gene sequence :M. lepraespecific u2266g 269 15539-15751 M. leprae gene sequence :M. lepraespecific u2266h 270 15996-15757 M. leprae gene sequence :M. lepraespecific u2266i 271 18690-17941 M. leprae gene sequence :M. lepraespecific u2266j 272 19344-18886 M. leprae gene sequence :M. lepraespeciflc u2266k 273 20639-20055 M. leprae gene sequence :M. lepraespecific u2266l 274 20752-20537 M. leprae gene sequence :M. lepraespeciflc u2266m 275 22137-21268 M. leprae gene sequence :M. lepraespecific u2266n 276 23030-23971 M. leprae gene sequence :M. lepraespecific u2266o 277 29564-28860 M. leprae gene sequence :M. lepraespecific u2266p 278 33161-32964 M. leprae gene sequence :M. lepraespecific u2266q 279 37362-36865 M. leprae gene sequence :M. lepraespecific u2266r 280 37443-37745 M. leprae gene sequence :M. lepraespecific u2266s 281 38205-38435 M. leprae gene sequence :M. lepraespecific u2266t 282 39609-39872 M. leprae gene sequence :M. lepraespecific

Mycobacterium Leprae peptides, the functions of such peptides, andnucleotide positions associated with such peptides within Seq. ID No.138 are summarized in Table XXIV below.

TABLE XXIV Name SeqID Position Enzyme or Protein Name Function 4c11 28332892-34358 4-coumarate-coA ligase :repair cut 284  9824-10657deoxyuridine 5′-triphosphate nucleotidohydrolase :metab:nt dcup 28523904-25109 uroporphyrinogen decarboxylase :synth hemy 286 24716-26461hemy protein :synth msg45mp 287 16345-15056 M. leprae gene sequence:transport myoP 288 7650-8537 myo-inositol-1(OR 4)-monophosphatase:antigen rps 289 6746-5004 DNA-directed RNA polymerase sigma:regulatory:transc tkt 290 16569-18965 transketolase :transport traA 29135961-34381 possible transcriptional activator (srmR) :regulatory u1764a292 1187-150  DNA-directed RNA polymerase :regulatory:transc u1764aa 29328189-27923 M. leprae gene sequence :M. leprae specific u1764ab 29428583-28398 M. leprae gene sequence :M. leprae specific u1764ac 29528952-29299 M. leprae gene sequence :M. leprae specific u1764ad 29630616-30837 M. leprae gene sequence :M. leprae specific u1764ae 29731124-31477 M. leprae gene sequence :M. leprae specific u1764af 29831478-31675 M. leprae gene sequence :M. leprae specific u1764b 2991776-1276 M. leprae gene sequence :M. leprae specific u1764c 3001687-2046 M. leprae gene sequence :M. leprae specific u1764d 3013055-2033 M. leprae gene sequence :M. leprae specific u1764e 3023610-3260 M. leprae gene sequence :M. leprae specific u1764f 3034856-4431 M. leprae gene sequence :M. leprae specific u1764fg 3048124-6739 M. leprae gene sequence :M. leprae specific u1764h 3059200-8541 M. leprae gene sequence :M. leprae specific u1764i 30610278-9694  M. leprae gene sequence :M. leprae specific u1764j 3079375-9689 M. leprae gene sequence :M. leprae specific u1764k 30810570-11475 M. leprae gene sequence :M. leprae specific u1764l 30912239-11520 M. leprae gene sequence :M. leprae specific u1764m 31012539-12772 M. leprae gene sequence :M. leprae specific u1764n 31113087-13278 M. leprae gene sequence :M. leprae specific u1764o 31214085-14282 M. leprae gene sequence :M. leprae specific u1764p 31314568-14756 M. leprae gene sequence :M. leprae specific u1764q 31416964-16398 M. leprae gene sequence :M. leprae specific u1764r 31518966-19160 M. leprae gene sequence :M. leprae specific u1764s 31619585-20049 M. leprae gene sequence :M. leprae specific u1764t 31720669-21169 M. leprae gene sequence :M. leprae specific u1764u 31822490-21066 M. leprae gene sequence :M. leprae specific u1764v 31922953-22357 M. leprae gene sequcnce :M. leprae specific u1764w 32023419-23063 M. leprae gene sequence :M. leprae specific u1764x 32123773-23468 M. leprae gene sequence :M. leprae specific u1764y 32226203-27162 M. leprae gene sequence :M. leprae specific u1764z 32327612-27869 M. leprae gene sequence :M. leprae specific

Mycobacterium Leprae peptides, the functions of such peptides, andnucleotide positions associated with such peptides within Seq. ID No.139 are summarized in Table XXV below.

TABLE XXV Name SeqID Position Enzyme or Protein Name Function achA 3242747-3214 acetyl-hydrolase :regulatory:transp cys 325 3622-5029 cysteinesynthase :synth:aa dhay 326 16015-15797 aldehyde dehydrogenase (NAD+):metab eutP1 327 15605-15339 ethanolamine permease (eutP) gene:transport eutP2 328 15289-15101 ethanolamine permease (eutP) gene:transport grea 329 7902-7237 transcription elongation factor GREA:regulatory:transc kre1 330 18977-19327 weak homolog of Saccharomycescerevisiae KRE1 :synth:cell wall lmbE 331 8458-9366 lmbE gene product:synth metB 332 6002-7174 cystathionine gamma-synthase :synth:aa prAG333 5140-5928 proline-rich antigen :antigen u1740 334 1795-1463 weakmatch to tyrocidine synthetase I :M. leprae specific u1740a 335  3-296M. leprae gene sequence :M. leprae specific u1740aa 336 29246-29563 M.leprae gene sequence :M. leprae specific u1740ab 337 29952-30137 M.leprae gene sequence :M. leprae specific u1740ac 338 30331-30693 M.leprae gene sequence :M. leprae speclfic u1740ad 339 30879-30550 M.leprae gene sequence :M. leprae specific u1740ae 340 30861-31070 M.leprae gene sequence :M. leprae specific u1740af 341 32594-33013 M.leprae gene sequence :M. leprae specific u1740ag 342 36000-36353 M.leprae gene sequence :M. leprae specific u1740ah 343 36801-36322 M.leprae gene sequence :M. leprae specific u1740ai 344 32476-32718 M.leprae gene sequence :M. leprae specific u1740aj 345 15049-14792 M.leprae gene sequence :M. leprae specific u1740b 346 9141-9668 M. lepraegene sequence :M. leprae specific u1740c 347 9502-9927 M. leprae genesequence :M. leprae specific u1740d 348  9824-10078 M. leprae genesequence :M. leprae specific u1740e 349 10059-10307 M. leprae genesequence :M. leprae seecific u1740f 350 10271-10705 M. leprae genesequence :M. leprae specific u1740g 351 11468-11716 M. leprae genesequence :M. leprae specific u1740h 352 12672-12361 M. leprae genesequence :M. leprae specific u1740i 353 14108-12684 lipoamidedehydrogenase :catab u1740j 354 14584-14219 M. leprae gene sequence :M.leprae specific u1740k 355 16311-15868 M. leprae gene sequence :M.leprae specific u1740l 356 16448-16176 aldehyde dehydrogenase :catab:aau1740m 357 17439-17735 M. leprae gene sequence :M. leprae specificu1740n 358 17701-17949 M. leprae gene sequence :M. leprae specificu1740o 359 18415-18858 prolyl endopeptidase :catab u1740p 36019727-19461 M. leprae gene sequence :M. leprae specific u1740q 36119621-20019 M. leprae gene sequence :M. leprae specific u1740r 36221077-20697 M. leprae gene sequence :M. leprae specific u1740s 36324193-21263 transport protein ;transport:antibiot u1740t 364 24672-24139M. leprae gene sequence :M. leprae specific u1740u 365 24695-24880 M.leprae gene sequence :M. leprae specific u1740v 366 24881-25093 M.leprae gene sequence :M. leprae specific u1740w 367 25762-25944 M.leprae gene sequence :M. leprae specific u1740x 368 26293-26742 M.leprae gene sequence :M. leprae specific u1740y 369 27179-28414transport protein ;transport:antibiot u1740z 370 28620-28907 M. lepraegene sequence :M. leprae specific

Mycobacterium Leprae peptides, the functions of such peptides, andnucleotide positions associated with such peptides within Seq. ID No.140 are summarized in Table XXVI below.

TABLE XXVI Name SeqID Position Enzyme or Protein Name Function accY 37132168-32404 weak match to acetyl-Co-Acarboxylase carboxyltrans. :metabatcB 372 27392-27045 calcium-transportin ATPase :transport atnA 37327907-27596 Na+, K+-ATPase alpha subunit :transport b650 374 19208-18924M. leprae gene sequence :M. leprae specific b650 375 19585-19325 M.leprae gene sequence :M. leprae specific phoP 376 35833-36063 alk phosphsyn transcriptional regulatory protein :regulalory:transc pstA 3771914-871  phosphate transport protein PSTA :transport pstC 378 2804-1803phosphate transport protein PSTC :transport pstS 379 3923-2805phosphate-repressible, periplasmic :transport smf2 380 6236-7522 SMF2protein :resistance znfy1 410 31567-32214 zinc finger protein:regulatory znfy2 411 31347-31709 zinc finger protein :regulatory u650381 24796-24281 M. leprae gene sequence :M. leprae specific u650 38227595-27386 M. leprae gene sequence :M. leprae specific u650a 3834393-4013 M. leprae gene sequence :M. leprae specific u650a 384 285-37 M. leprae gene sequence :M. leprae specific u650ab 385 32699-32932 M.leprae gene sequence :M. leprae specific u650ac 386 33365-33613 M.leprae gene sequence :M. leprae specific u650b 387 4523-4840 M. lepraegene sequence :M. leprae specific u650e 388 5417-5632 M. leprae genesequence :M. leprae specific u650f 389 8177-7995 M. leprae gene sequence:M. leprae specific u650g 390 8860-8624 M. leprae gene sequence :M.leprae specific u650h 391 10471-10154 M. leprae gene sequence :M. lepraespecific u650i 392 10711-10472 M. leprae gene sequence :M. lepraespecific u650j 393 10880-11248 M. leprae gene sequence :M. lepraespecific u650k 394 12043-12450 M. leprae gene sequence :M. lepraespecific u650l 395 13094-12894 M. leprae gene sequence :M. lepraespecific u650m 396 14944-15813 M. leprae gene sequence :M. lepraespecific u650n 397 15878-16153 M. leprae gene sequence :M. lepraespecific u650o 398 16958-16734 M. leprae gene sequence :M. lepraespecific u650p 399 18652-18467 M. leprae gene sequence :M. lepraespecific u650q 400 20450-20656 M. leprae gene sequence :M. lepraespecific u650r 401 22198-22539 M. leprae gene sequence :M. lepraespecific u650s 402 22591-22776 M. leprae gene sequence :M. lepraespecific u650t 403 22777-22959 lignostilbene alphabeta-dioxygenase:catab u650u 404 25611-25315 M. leprae gene sequence :M. leprae specificu650v 405 26166-25612 M. leprae gene sequence :M. leprae specific u650w406 28135-27908 M. leprae gene sequence :M. leprae specific u650x 40728993-28640 M. leprae gene sequence :M. leprae specific u650y 40829213-28989 M. leprae gene sequence :M. leprae specific u650z 40931263-30511 4-chlorobenzoate-CoA dehalogenase :snyth:lipid dnfa 41031567-32214 zinc finger protein :regulatory dnfb 411 31347-31709 zincfinger protein :regulatory

Proteins and peptides of the present invention have further utility foruse as vaccines or as screens for new tuberculosis drugs. The purifiedproteins derived from Mycobacterium tuberculosis or leprae may elicit aspecific immune response. The production of purified proteins byrecombinant means is not limited to the use of pathogenic bacteria. Theproteins of this invention, derived from M. tuberculosis or M. leprae,may also be expressed in M. bovis-BCG by recombinant means, and theresulting recombinant BCG cells may be used as a vaccine. See publishedPCT application WO90/00594, for example.

The availability of large quantities of Mycobacterium tuberculosisand/or leprae proteins and peptides allows the use of such proteins andpeptides as specific binding agents for drugs. The proteins and peptidesmay also be used to elicit immune responses for the development ofantibodies. Such antibodies may have therapeutic value. Proteins andpeptides may also be used diagnostically to detect hypersensitivityreactions of individuals exposed to Mycobacterium tuberculosis orleprae. The proteins and peptides may be affixed to solid supports todetect antibodies from patient's sera typical of hypersensitivityreactions.

For example, one method comprises the step of forming an admixture of asera of an individual with a peptide corresponding to a peptide ofMycobacterium tuberculosis or leprae. Upon imposition of reactionconditions in the presence of an antibody to the peptide, an affinitycomplex forms. The method further includes the step of detecting theaffinity complex which affinity complex is indicative of the presence ofa hypersensitivity reaction.

One embodiment of the present invention features, as an article ofmanufacture, a kit comprising a non-naturally occurring nucleic acidcorresponding to Mycobacterium tuberculosis or leprae nucleic acid. Afurther embodiment of the present invention features a kit comprising anon-naturally occurring peptide corresponding to a peptide ofMycobacterium tuberculosis or leprae or an antibody capable of bindingsuch peptides.

These and other features will be apparent to individuals skilled in theart upon reading the detailed description of the present invention.

DETAILED DECSRIPTION

The present invention relates to nucleic acid and amino acid sequencesrelating to Mycobacterium tuberculosis and leprae. As used herein, theterm “Mycobacterium tuberculosis” is used in the sense of the causativeagent of tuberculosis in humans. The term “Mycobacterium leprae” refersto the causative agent of leprosy in humans.

As used herein the term “corresponding to”, referring to a nucleic acid,means nucleotides in a sequence which is homologous or complementary toother nucleic acid or with reference to a peptide, homologous orcomplementary to the nucleic acid which encodes such peptides. Thederived nucleic acid may be generated in any manner, including, forexample, chemical synthesis, or DNA polymerase or reverse transcriptionwhich are based on the sequence information.

Similarly, the term “corresponding to”, referring to a peptide or aprotein, means having an amino acid sequence encoded by a designatednucleic acid sequence, or which is immunologically identical with apeptide encoded in the sequence, or having the amino acid sequence of adesignated peptide. The peptide encoded by the sequence is the geneproduct. A corresponding peptide is not necessarily translated from acorresponding nucleic acid sequence but may be generated in any manner,including, for example, chemical synthesis, or expression of arecombinant expression system or isolation from mutated Mycobacteriumtuberculosis or leprae.

The term “non-naturally occurring nucleic acid” comprises genomic,synthetic DNA, semi-synthetic, or a synthetic nucleic acid which byvirtue of its origin or manipulation is not associated with all or aportion of the nucleic acid with which it is associated in nature,and/or in a form of a library, and/or is linked to a nucleic acid otherthan that to which it is linked in nature. The term “associated with” isused to denote linkage such as that between adjacent segments of nucleicacid.

“Host cells” and other such terms denoting micro-organisms or highereukaryotic cell lines cultured as unicellular entities refers to cellswhich can become or have been used as recipients for a recombinantvector or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood by individuals skilledin the art that the progeny of a single parental cell may notnecessarily be completely identical in genomic or total DNA complimentto the original parent, due to accident or deliberate mutation.

The term “control sequence” refers to a nucleic acid having a basesequence which is recognized by the host organism to effect theexpression of encoded sequences to which they are ligated. The nature ofsuch control sequences differs depending upon the host organism; inprokaryotes, such control sequences generally include a promoter,ribosomal binding site and terminators; in eukaryotes, generally suchcontrol sequences include promoters, terminators and in some instances,enhancers. The term control sequence is intended to include at aminimum, all components whose presence is necessary for expression, andmay also include additional components whose presence is advantageous,for example, leader sequences.

The term “operably linked” refers to sequences joined or ligated tofunction in their intended manner. For example, a control sequence isoperably linked to coding sequence by ligation in such a way thatexpression of the coding sequence is achieved under conditionscompatible with the control sequence and lost cell.

An “open reading frame” is a region of nucleic acid which encodes apeptide. This region may represent a portion of a coding sequence or atotal sequence.

A “coding sequence” is a nucleic acid sequence which is transcribed intomessenger RNA and/or translated into a peptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start code at the fiveprime terminus and a translation stop code at the three prime terminus.A coding sequence can include but is not limited to messenger RNA,synthetic DNA, and recombinant nucleic acid sequences.

A “gene product” is a protein or structural RNA which is specificallyencoded for by a gene.

The term “probe” refers to a nucleic acid, peptide or other chemicalentity which specifically binds to a molecule of interest. Probes areoften associated with or capable of associating with a label. A label isa chemical moiety capable of detection. Typical labels comprise dyes,radioisotopes, luminescent and chemiluminescent moieties, fluorophores,enzymes, precipitating agents, amplification sequences, and the like.Similarly, a nucleic acid, peptide or other chemical entity whichspecifically binds to a molecule of interest and immobilizes suchmolecule is referred herein as a “capture ligand”. Capture ligands aretypically associated with or capable of associating with a support suchas nitro-cellulose, glass, nylon membranes, beads, particles and thelike. The specificity of hybridization is dependent on conditions suchas the base pair composition of the nucleotides, and the temperature andsalt concentration of the reaction. These conditions are readilydiscernable to one of ordinary skill in the art using routineexperimentation. The experimental manipulation of such conditions hasbeen well described in the literature including such books as MolecularCloning; A Laboratory Manual, Sambrook, J., Fritsch, E. F., Maniatis,T., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2nd ed.(1989).

The term “primer” is used to denote nucleic acid capable of binding to aspecific sequence and initiating a polymerase reaction.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, and immunology, which are within theskill of the art. Such techniques are explained fully in the literature.See e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning; ALaboratory Manual 2nd ed. (1989); DNA Cloning, Volumes I and II (D. NGlover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984);Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); theseries, Methods in Enzymology (Academic Press, Inc.), particularly Vol.154 and Vol. 155 (Wu and Grossman, eds.) and PCR-A Practical Approach(McPherson, Quirke, and Taylor, eds., 1991).

The DNA sequences relating to Mycobacterium tuberculosis are derivedfrom nucleic acid sequences present in Mycobacterium tuberculosis.Mycobacterium tuberculosis from which the sequences are derived wasacquired from the American Type Culture Collection and has an ATCCdesignation 25618. This particular Mycobacterium tuberculosis hasfeatures which are consistent with those described of Mycobacteriumtuberculosis in general and is believed to be representative of allMycobacterium tuberculosis. The Mycobacterium tuberculosis of thedeposit is available through the catalogue of the American Type CultureCollection without restriction. It is believed the Mycobacteriumtuberculosis of the deposit has been and is widely available.

The nucleic acid sequences of this invention may be obtained directlyfrom the DNA of the above referenced Mycobacteria tuberculosis strain byusing the polymerase chain reaction (PCR). See “PCR, A PracticalApproach” (McPherson, Quirke, and Taylor, eds., IRL Press, Oxford, UK,1991) for details about the PCR. Alternatively, the sequences of thisinvention may be obtained from libraries of Mycobacteria DNA fragmentscarried in clones of suitable host organisms such as E. coli. Suitablelibraries include those of Eiglmeier et al. (1993, Mol. Microbiol. 7,197-206) and Clark-Curtiss et al. (1985, J. Bacteriol. 161, 1093-1102)for M. leprae and those of Kalpana et al. (1991, Proc. Natl. Acad. Sci.USA 88, 5433-5437) and Bhargava et al. (1990, J. Bacteriol. 172,2930-2934) for M. tuberculosis. Clones carrying the desired sequencesdescribed in this invention may be obtained by screening the librariesby means of the PCR or by hybridization of synthetic oligonucleotideprobes to filter lifts of the library colonies or plaques as known inthe art (see, eg, Sambrook et al., “Molecular Cloning, A LaboratoryManual” 2nd edition, 1989, Cold Spring Harbor Press, NY).

Nucleic acids isolated or synthesized in accordance with features of thepresent invention are useful, by way of example, without limitation, asprobes, primers, capture ligands, anti-sense genes and for developingexpression systems for the synthesis of proteins and peptidescorresponding to such sequences.

As probes, primers, capture ligands and anti-sense agents, the nucleicacid will normally comprise approximately twenty or more nucleotides forspecificity as well as the ability to form stable hybridizationproducts.

Probes

A nucleic acid isolated or synthesized in accordance with Seq. ID No. 1or Seq. ID Nos. 23-26 and 120-140 can be used as a probe to specificallydetect Mycobacterium tuberculosis or M. leprae, respectively. With thesequence information set forth in the present application, sequences oftwenty or more nucleotides are identified which provide the desiredinclusivity and exclusivity with respect to Mycobacterium tuberculosisand/or M. leprae, and extraneous nucleic acid sequences likely to beencountered during hybridization conditions. More preferably, thesequence will comprise at least twenty to thirty nucleotides to conveystability to the hybridization product formed between the probe and theintended target molecules.

Sequences larger than 1000 nucleotides in length are difficult tosynthesize but can be generated by recombinant DNA techniques.Individuals skilled in the art will readily recognize that the nucleicacid sequences, for use as probes, can be provided with a label tofacilitate detection of a hybridization product.

Nucleic acid isolated and synthesized in accordance with Seq. ID No. 1or Seq. ID Nos. 23-26 and 120-140 may also be useful as probes to detecthomologous regions (especially homologous genes) of M. tuberculosis, M.leprae, M. avium, M. bovis, or other mycobacterial species using relaxedstringency hybridization conditions, as will be obvious to anybodyskilled in the art.

Capture Ligand

For use as a capture ligand, the nucleic acid selected in the mannerdescribed above with respect to probes, can be readily associated with asupport. The manner in which nucleic acid is associated with supports iswell known. Nucleic acid having twenty or more nucleotides in a sequencecorresponding to Seq. ID No. 1 with respect to Mycobacteriumtuberculosis, or Seq. ID Nos. 23-26 and 120-140 with respect toMycobacterium M. leprae have utility to separate Mycobacteriumtuberculosis or leprae nucleic acid from the nucleic acid of each otherand other organisms. Nucleic acid having twenty or more nucleotides in asequence corresponding to Seq. ID Nos. 1 and 23-26 and 120-140 may alsohave utility to separate M. avium or M. bovis or other mycobacterialspecies from each other and from other organisms. Preferably, thesequence will comprise at least twenty nucleotides to convey stabilityto the hybridization product formed between the probe and the intendedtarget molecules. Sequences larger than 1000 nucleotides in length aredifficult to synthesize but can be generated by recombinant DNAtechniques.

Primers

Nucleic acid isolated or synthesized in accordance with the sequencesdescribed herein have utility as primers for the amplification ofMycobacterium tuberculosis and M. leprae nucleic acid. These nucleicacids may also have utility as primers for the amplification of nucleicacid sequences in M. avium, M. bovis, or other mycobacterial species.With respect to polymerase chain reaction (PCR) techniques, nucleic acidsequences of twenty or more nucleotides corresponding to Seq. ID No. 1or Seq. ID Nos. 23-26 and 120-140 have utility in conjunction withsuitable enzymes and reagents to create copies of Mycobacteriumtuberculosis and/or leprae nucleic acid. More preferably, the sequencewill comprise twenty to thirty nucleotides to convey stability to thehybridization product formed between the probe and the intended targetmolecules. Sequences larger than 1000 nucleotides in length aredifficult to synthesize but can be generated by recombinant DNAtechniques. Binding conditions of probes greater than 100 nucleotidesare more difficult to control to obtain specificity.

The copies can be used in diagnostic assays to detect specificsequences, including genes Mycobacterium tuberculosis and/or M. lepraeand/or M. avium, M. bovis, or other mycobacteria species. The copies canalso be incorporated into cloning and expression vectors to generatepolypeptides corresponding to the nucleic acid synthesized by PCR, aswill be described in greater detail below.

Anti-sense

Nucleic acid or nucleic acid-hybridizing derivatives isolated orsynthesized in accordance with the sequences described herein haveutility as anti-sense agents to prevent the expression of Mycobacteriumtuberculosis or M. leprae genes. These sequences may also have utilityas anti-sense agents to prevent expression of genes of M. avium, M.bovis, or other mycobacteria species.

Nucleic acid or derivatives corresponding to Mycobacterium tuberculosisor M. leprae nucleic acid sequences is loaded into a suitable carriersuch as a liposome or mycobacteriaphage for introduction into amycobacterial cells. For example, a nucleic acid having twenty or morenucleotides is capable of binding to bacteria nucleic acid or bacteriamessenger RNA. Preferably, the anti-sense nucleic acid is comprised of20 or more nucleotides to provide necessary stability of a hybridizationproduct of non-naturally occurring nucleic acid and bacterial nucleicacid and/or bacterial messenger RNA. Nucleic acid having a sequencegreater than 1000 nucleotides in length is difficult to synthesize butcan be generated by recombinant DNA techniques. Methods for loadinganti-sense nucleic acid in liposomes is known in the art as exemplifiedby U.S. Pat. No. 4,241,046 issued Dec. 23, 1980 to Papahadjopoulos etal.

Expressing Mycobacterial Genes

The function of a specific gene or operon can be ascertained byexpression in a bacterial strain under conditions where the activity ofthe gene product(s) specified by the gene or operon in question can bespecifically measured. Alternatively, a gene product may be produced inlarge quantities in an expressing strain for use as an antigen, anindustrial reagent, for structural studies, etc. This expression couldbe accomplished in a mutant strain which lacks the activity of the geneto be tested, or in a strain that does not produce the same geneproduct(s). This includes, but is not limited to, mycobacterial strainssuch as BCG and M. Smegmatis, and other bacterial strains such as E.coli, Norcardia, Corynebacterium, and Streptomyces species. In somecases the expression host will utilize the natural mycobacterialpromoter whereas in others, it will be necessary to drive the gene witha promoter sequence derived from the expressing organism (e.g., an E.coli beta-galactosidase promoter for expression in E. coli).

To express a gene product using the natural mycobacterial promoter, aprocedure such as the following is used. A restriction fragmentcontaining the gene of interest, together with its associated naturalpromoter elements and regulatory sequences (identified using the DNAsequence data) is cloned into an appropriate recombinant plasmidcontaining the following components: an origin of replication thatfunctions in the host organism, and an appropriate selectable marker.This can be accomplished by a number of procedures known to thoseskilled in the art. It is most preferably done by cutting the plasmidand the fragment to be cloned with the same restriction enzyme toproduce compatible ends that can be ligated to join the two piecestogether. The recombinant plasmid is introduced into the host organismby electroporation and cells containing the recombinant plasmid areidentified by selection for the marker on the plasmid. Expression of thedesired gene product is detected using an assay specific for that geneproduct.

In the case of a gene that requires a different promoter, the body ofthe gene (coding sequence) is specifically excised and cloned into anappropriate expression plasmid. This subcloning can be done by severalmethods, but is most easily accomplished by PCR amplification of aspecific fragment and ligation into to expression plasmid after treatingthe PCR product with a restriction enzyme or exonuclease to createsuitable ends for cloning.

Expressed Genes in Therapeutics

Nucleic acid isolated or synthesized in accordance with the sequencesdescribed herein have utility to generate proteins and peptides. Thenucleic acid exemplified by Seq. ID No. 1 or Seq. ID No. 23-26 and120-140 can be cloned into suitable vectors or used to isolate nucleicacid. The isolated nucleic acid is combined with suitable DNA linkersand cloned into a suitable vector. The vector can be used to transform asuitable host organism such as E. coli and the peptide or proteinencoded by the sequences isolated.

Molecular cloning techniques are described in the text MolecularCloning: A Laboratory Manual, 2nd edition, Sambrook et al., ColdspringHarbor Laboratory (1989). The isolated peptide has utility as anantigenic substance for the development of vaccines and antibodiesdirected to Mycobacterium tuberculosis or M. leprae.

The isolated protein or peptide also has utility in screening assays toidentify inhibitors or potentiators of the activity of said protein orpeptide. Such inhibitors or potentiators may be useful as newtherapeutic agents to combat Mycobacterial infections in man. Screeningassays may be constructed in vitro with purified Mycobacterial enzymesuch that the action of the enzyme produces an easily detectablereaction product. Suitable products include those with distinctiveabsorption, fluorescence, or chemi-luminescence properties, for example,because detection may be easily automated. A variety of synthetic ornaturally occurring compounds may be tested in the assay to identifythose which inhibit or potentiate the activity of the mycobacterialenzyme. Some of these active compounds may directly, or with chemicalalterations to promote membrane permeability or solubility, also inhibitor potentiate the same enzymatic activity in whole, live mycobacterialcells. Such compounds may be used as anti-mycobacterials in therapy.Since cells of M. tuberculosis and M. leprae grow poorly or not at allin culture, in vitro assays with isolated M. tuberculosis or M. lepraeenzymes provide a practical approach for detecting potential newtherapeutic agents.

Alternatively, new therapeutic agents may be discovered by use ofscreening assays which incorporate the M. tuberculosis or leprae genesof this invention or fragments thereof into other micro-organisms suchas other Mycobacteria species. For example, M. tuberculosis or M. lepraegenes expressed in heterologous micro-organisms may create new productswhose activity or presence can be assayed. Agents which alter theactivity or presence of these products may be useful therapeutic agentsto combat M. tuberculosis or M. leprae infections in man. This approachis useful even when the function or activity of the protein or enzymeencoded by the gene is poorly understood or difficult to assay.Preferably, the genes are essential for growth or viability of theorganism. Mycobacteria smegmatis and M. bovis are non-pathogenic but areclosely related to M. tuberculosis and M. leprae and are much easier togrow and manipulate in culture. Molecular genetic methods permit thetransformation of M. bovis and M. smegmatis with autonomouslyreplicating vectors and with integrating vectors capable of carrying newgenes. See, for example, details provided in published PCT applicationsWO 88/06626, WO 90/00594, WO 92/01783, WO 92/01796, and WO 92/22326.

This methodology may be used to replace M. bovis or M. smegmatis geneswith their M. tuberculosis or M. leprae homologs provided in thisinvention. For example, the M. bovis or M. smegmatis gene which ishomologous to the M. tuberculosis or M. leprae gene of interest isidentified from appropriate clone libraries (see, for example, Kalpanaet al., 1991, Proc. Natl. Acad. Sci. USA 88, 5433-5437) by colony orplaque hybridization using the M. tuberculosis or M. leprae genes ofthis invention or fragments thereof as probes. The resulting M. bovis orM. smegmatis gene is rendered non-functional by disruption or deletionof the coding region and re-introduced into M. bovis or M. smegmatis byhomologous recombination as a linear fragment (see Kalpana et al., 1991,above). Homologous replacements may be distinguished from the backgroundof illegitimate integrations-by using the PCR with appropriate primers.Nested PCR primers may be used in a two-stage PCR to increasespecificity if necessary as is known in the art. Clones carrying thereplacement will be inviable if the gene is essential. However, thereplacement may also be performed simultaneously with clones carryingthe homologous M. tuberculosis or leprae gene either on an autonomouslyreplicating plasmid or integrated elsewhere in the genome. If the M.tuberculosis or leprae gene complements the function of the homologousM. bovis or M. smegmatis gene, then the resulting replacement cloneswill be viable.

Resulting clones carrying specific M. tuberculosis or leprae genes asfunctional replacements for the homologous M. bovis or smegmatis genesare useful in in vivo screens for new therapeutic agents. Specifically,synthetic or naturally occurring compounds may be applied to cultures ofthese functional replacement clones and simultaneously to cultures ofunmodified M. bovis or smegmatis. Compounds which inhibit measurableproperties such as the growth or viability of the functional replacementclones significantly more effectively than they inhibit the unmodifiedcontrol clones may be useful therapeutic agents for specific treatmentof M. tuberculosis or leprae infections in man.

Expressed Genes in Vaccines

The peptide materials of the present invention have utility for thedevelopment of antibodies and vaccines.

The availability of nucleotide sequences derived from Mycobacteriumtuberculosis and/or leprae (including segments and modifications of thesequence), permits the construction of expression vectors encodingantigenically active regions of a peptide.

Such expression vectors may be introduced into mycobacteria such as M.bovis-BCG to provide stable production of the desired peptide by therecombinant mycobacteria cells. See, for example, details of theexpression method provided in published PCT applications WO 88/06626, WO90/00594, and WO 92/01796. In this manner, the excellent adjuvantproperties of the mycobacteria are used to full advantage for theproduction of M. tuberculosis or M. leprae vaccines. Alternatively, thepeptides may be produced by recombinant means in a variety of host cellsincluding, but not limited to, E. coli and subsequently purified for usein vaccines and diagnostic tests.

Fragments encoding the desired peptides are derived from the clonesusing conventional restriction digestion or by synthetic methods, andare ligated into vectors which may, for example, contain portions offusion sequences such as beta galactosidase or superoxide dismutase(SOD), preferably SOD. Methods and vectors which are useful for theproduction of polypeptides which contain fusion sequences of SOD aredescribed in European Patent Office Publication number 0196056,published Oct. 1, 1986.

Any desired portion of the Seq. ID No. 1 or Seq. ID Nos. 23-26 and120-140 containing an open reading frame, can be obtained as arecombinant peptide, such as a mature or fusion protein; alternatively,a peptide encoded in an open reading frame can be provided by chemicalsynthesis.

The DNA encoding the desired peptide, whether in fused or mature form,and whether or not containing a signal sequence to permit secretion, maybe ligated into expression vectors suitable for any convenient host.Both eukaryotic and prokaryotic host systems are presently used informing recombinant peptides. The peptide is then isolated from lysedcells or from the culture medium and purified to the extent needed forits intended use. Purification may be by techniques known in the art,for example, differential extraction, salt fractionation, chromatographyon ion exchange resins, affinity chromatography, centrifugation, and thelike. See, for example, Methods in Enzymology, supra, for a variety ofmethods for purifying proteins. Such peptides can be used asdiagnostics, or those which give rise to neutralizing antibodies may beformulated into vaccines. Antibodies raised against these peptides canalso be used as diagnostics, or for passive immunotherapy or forisolating and identifying Mycobacterium tuberculosis or leprae.

An antigenic region of a peptide is generally relatively small—typically8 to 10 amino acids or less in length. Fragments of as few as 5 aminoacids may characterize an antigenic region. Accordingly, DNAs encodingshort segments of Mycobacterium tuberculosis or leprae peptides can beexpressed recombinantly either as fusion proteins, or as isolatedpeptides. In addition, short amino acid sequences can be convenientlyobtained by chemical synthesis. In instances wherein the synthesizedpeptide is correctly configured so as to provide the correct epitope,but is too small to be immunogenic, the peptide may be linked to asuitable carrier.

A number of techniques for obtaining such linkage are known in the art,including the formation of disulfide linkages usingN-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and succinimidyl4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC) obtained fromPierce Company, Rockford, Ill., (if the peptide lacks a sulfhydrylgroup, this can be provided by addition of a cysteine residue). Thesereagents create a disulfide linkage between themselves and peptidecysteine residues on one protein and an amide linkage through theepsilon-amino on a lysine, or other free amino group in the other. Avariety of such disulfide/amide-forming agents are known. See, forexample, Immun Rev (1982) 62:185. Other bifunctional coupling agentsform a thioether rather than a disulfide linkage. Many of thesethio-ether-forming agents are commercially available and includereactive esters of 6-maleimidocaprioc acid, 2-bromoacetic acid,2-iodoacetic acid, 4-N-maleimido-methyl)cyclohexane-1-carboxylic acid,and the like. The carboxyl groups can be activated by combining themwith succinimide or 1-hydroxyl-2 nitro-4-sulfonic acid, sodium salt.Additional methods of coupling antigens employ the rotavirus “bindingpeptide” system described in EPO Pub. No. 259,149, the disclosure ofwhich is incorporated herein by reference. The foregoing list is notmeant to be exhaustive, and modifications of the named compounds canclearly be used.

Any carrier may be used which does not itself induce the production ofantibodies harmful to the host. Suitable carriers are typically large,slowly metabolized macromolecules such as proteins; polysaccharides,such as latex functionalized sepharose, agarose, cellulose, cellulosebeads and the like; polymeric amino acids, such as polyglutamic acid,polylysine, and the like; amino acid copolymers; and inactive virusparticles. Especially useful protein substrates are serum albumins,keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin,ovalbumin, tetanus toxoid, and other proteins well known to thoseskilled in the art.

Peptides comprising Mycobacterium tuberculosis or leprae amino acidsequences encoding at least one epitope are useful immunologicalreagents. For example, peptides comprising such truncated sequences canbe used as reagents in an immunoassay. These peptides also are candidatesubunit antigens in compositions for antiserum production or vaccines.While the truncated sequences can be produced by various knowntreatments of native Mycobacterium tuberculosis or leprae protein, it isgenerally preferred to make synthetic or recombinant peptides. Peptidescomprising these truncated sequences can be made up entirely ofMycobacterium tuberculosis or leprae sequences (one or more epitopes,either contiguous or noncontiguous), or Mycobacterium tuberculosis orleprae sequences and heterologous sequences in a fusion protein. Usefulheterologous sequences include sequences that provide for secretion froma recombinant host, enhance the immunological reactivity of theepitope(s), or facilitate the coupling of the polypeptide to animmunoassay support or a vaccine carrier. See, E.G., EPO Pub. No.116,201; U.S. Pat. No. 4,722,840; EPO Pub. No. 259,149; U.S. Pat. No.4,629,783.

The size of peptides comprising the truncated sequences can vary widely,the minimum size being a sequence of sufficient size to provide anepitope, while the maximum size is not critical. For convenience, themaximum size usually is not substantially greater than that required toprovide the desired epitope and function(s) of the heterologoussequence, if any. Typically, the truncated amino acid sequence willrange from about 5 to about 100 amino acids in length. More typically,however, the sequence will be a maximum of about 50 amino acids inlength, preferably a maximum of about 30 amino acids. It is usuallydesirable to select sequences of at least about 10, 12 or 15 aminoacids, up to a maximum of about 20 or 25 amino acids.

Mycobacterium tuberculosis or leprae amino acid sequences comprisingepitopes can be identified in a number of ways. For example, the entireprotein sequence corresponding to each open reading frame is screened bypreparing a series of short peptides that together span the entireprotein sequence of such open reading frame. By starting with, forexample, peptides of approximately 100 amino acids, it would be routineto test each peptide for the presence of epitope(s) showing a desiredreactivity, and then testing progressively smaller and overlappingfragments from an identified peptides of 100 amino acids to map theepitope of interest. Screening such peptides in an immunoassay is withinthe skill of the art. It is also known to carry out a computer analysisof a protein sequence to identify potential epitopes, and then preparepeptides comprising the identified regions for screening.

The immunogenicity of the epitopes of Mycobacterium tuberculosis and/orleprae may also be enhanced by preparing them in mammalian or yeastsystems fused with or assembled with particle-forming proteins.Constructs wherein the Mycobacterium tuberculosis and/or leprae epitopeis linked directly to the particle-forming protein coding sequencesproduce hybrids which are immunogenic with respect to the Mycobacteriumtuberculosis and/or leprae epitope.

Vaccines

Vaccines may be prepared from one or more immunogenic peptides derivedfrom Mycobacterium tuberculosis or leprae. The observed homology betweenMycobacterium tuberculosis and/or leprae with other bacteria providesinformation concerning the peptides which are likely to be mosteffective as vaccines, as well as the regions of the genome in whichthey are encoded. Multivalent vaccines against Mycobacteriumtuberculosis or leprae may be comprised of one or more epitopes from oneor more proteins.

The preparation of vaccines which contain an immunogenic peptide as anactive ingredient, is known to one skilled in the art. Typically, suchvaccines are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified, or the protein encapsulated in liposomes. The activeimmunogenic ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and/or adjuvantswhich enhance the effectiveness of the vaccine. Examples of adjuvantswhich may be effective include but are not limited to: aluminumhydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1-2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against an immunogenicpeptide containing an HCV antigenic sequence resulting fromadministration of this peptide in vaccines which are also comprised ofthe various adjuvants.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0/5% to 10%, preferably 1%-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like.

Kits

The nucleic acid, peptides and antibodies of the present invention canbe combined with other reagents and articles to form kits. Kits fordiagnostic purposes typically comprise the nucleic acid, peptides orantibodies in vials or other suitable vessels. Kits typically compriseother reagents for performing hybridization reactions, polymerase chainreactions (PCR), or for reconstitution of lyophilized components, suchas aqueous media, salts, buffers, and the like. Kits may also comprisereagents for sample processing such as detergents, chaotropic salts andthe like. Kits may also comprise immobilization means such as particles,supports, wells, dipsticks and the like. Kits may also comprise labelingmeans such as dyes, developing reagents, radioisotopes, fluorescentagents, luminescent or chemiluminescent agents, enzymes, intercalatingagents and the like. With the nucleic acid and amino acid sequenceinformation provided herein, individuals skilled in art can readilyassemble kits to serve their particular purpose.

Embodiments of the present invention are further described with respectto the sequence listings which follow.

SEQUENCE LISTING The patent contains a lengthy “Sequence Listing”section. A copy of the “Sequence Listing” is available in electronicform from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=06583266B1). An electroniccopy of the “Sequence Listing” will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A non-naturally occurring peptide ofMycobacterium tuberculosis comprising an amino acid sequencecorresponding to the amino acid sequence of SEQ ID NO:12.